Mechanochemical control of epidermal stem cell divisions by B-plexins

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

D uring embryogenesis, tissue growth and shape are intimately connected 1 . Tissue growth intrinsically generates mechanical strains and compressions, and, conversely, mechanical deformations provide a fundamental regulatory feedback for growth control [1][2][3] . This feedback requires cells to sense mechanical forces, which are then transformed into biochemical signals that in turn control cellular functions including the rate of cell divisions [3][4][5][6] . Compression of cells within a tissue results in suppression of proliferation, a response termed contact inhibition of proliferation 2,7 . In the mammalian embryonic epidermis, proliferation of stem cells within the mechanically jammed basal layer causes crowding and local cell stress anisotropy [8][9][10] . How epidermal stem cells sense these mechanical forces and couple them with division is largely unknown.
Plexins constitute a family of single-pass transmembrane proteins that have initially been described for their role as axon guidance receptors for semaphorins in the developing nervous system 11,12 . In vertebrates, nine plexins have been identified, which-based on homology-are grouped into four subfamilies, Plexin-A1-4, Plexin-B1-3, Plexin-C1, and Plexin-D1 13 . They are now recognized to be of central importance for cell-cell communication in multiple tissues and biological systems, such as the immune and bone system, as well as in cancer [14][15][16] . Recently, Plexin-D1 has been shown to be activated by fluid shear stressinduced mechanical forces in vascular endothelial cells 17 . Whether plexins play a role in mechanosensation in epithelial cells is unknown.
Here, we report that epidermal stem cells require Plexin-B1 and Plexin-B2 to sense mechanical compression, and uncover Plexin-B1 and Plexin-B2 as negative upstream regulators of YAP that suppress stem cell divisions in response to crowding. This biomechanical feedback mechanism ensures a robust control of proliferation rates by cell density, and is functionally relevant for both tissue morphogenesis as well as cancer.

Results
Plexin-B1/Plexin-B2 suppress epidermal stem cell proliferation during embryonic development. Divisions of stem cells within an epithelial layer induce crowding and cell shape anisotropy 18 . We hypothesized that a mechanical feedback to control stem cell divisions would therefore be particularly relevant during developmental stages with high stem cell proliferation rates. We observed that in the developing mouse epidermis, stem cell proliferation rates were highest at embryonic day 15.5 (E15.5) and gradually declined to very low rates in the adult (Fig. 1a, b), consistent with previous reports 19,20 . The mRNA expression levels of plexins showed a striking positive correlation with proliferation rates, with Plexin-B2 being the most highly expressed plexin family member during embryonic development ( Fig. 1c and Supplementary Fig. 1a, b). Immunostainings revealed that Plexin-B2 was expressed in all layers of the developing epidermis including the K14-positive stem cells (Fig. 1d, e). Plexin-B1, a receptor highly homologous to Plexin-B2 11,13 , localized exclusively to the stem cell layer (Fig. 1d). To identify the functional role of Plexin-B1 and Plexin-B2 in epidermal stem cell proliferation, we inactivated the respective genes. Given (1) the overlapping expression pattern of Plexin-B1 and Plexin-B2 in K14-positive stem cells of the epidermis (Fig. 1d), (2) the functional redundancy of these two receptors during development of other tissues [21][22][23] , and (3) our observation that Plexin-B1 singledeficient and Plexin-B2 single-deficient embryos were devoid of any epidermal abnormalities ( Supplementary Fig. 1c-f), we generated mice lacking both Plexin-B1 and Plexin-B2 in epidermal stem cells by crossing conditional alleles for Plexin-B1 and Plexin-B2 with a constitutively expressed keratin 14-Cre line (K14-Cre;plxnb1 flox/flox ;plxnb2 flox/flox ). Consistent with the onset of Cre expression 24 , Plexin-B1 and Plexin-B2 expression was lost as early as E15.5 ( Supplementary Fig. 1g). Interestingly, Plexin-B1/Plexin-B2 double-knockout mice ("PlexDKO") showed a marked epidermal hyperproliferation (Fig. 1f, g), resulting in epidermal thickening (Fig. 1h, i). This epidermal thickening was due to an expansion of K14-positive stem cells, while the number of K10-positive differentiated cells was not significantly increased (Fig. 1j, k and Supplementary 1h). Apoptosis rates remained unchanged ( Supplementary Fig. 1i). Taken together, these results indicate that Plexin-B1 and Plexin-B2 suppress epidermal stem cell proliferation during embryonic development.
Hyperproliferation of Plexin-B1/Plexin-B2 double-deficient epidermal stem cells induces overcrowding, cell shape anisotropy and differentiation. We next investigated how the increased number of epidermal stem cell divisions in Plexin-B1/ Plexin-B2 double-knockout mice affected stem cell density and shape. To do so, we performed segmentation analyses of the epidermal stem cell layer at embryonic day 15.5 (Fig. 2a). In mice lacking Plexin-B1 and Plexin-B2, we observed both a higher cell density (Fig. 2b) as well as smaller and more variable cell sizes ( Fig. 2c) than in control mice. This was accompanied by an increase in cell shape anisotropy, with cells being more elongated (Fig. 2d, e). Similar abnormalities were detected at embryonic day 16.5 ( Supplementary Fig. 2a-e). These data demonstrate that Plexin-B1/Plexin-B2 double-deficiency results in overcrowding and shape deformation in the epidermal stem cell layer.
It is known that proliferation-induced crowding and cell shape distortion trigger differentiation and delamination of epidermal stem cells 18 . To assess whether loss of Plexin-B1/Plexin-B2 affects stem cell differentiation, we performed immunostainings for active Notch (Notch intracellular domain (NICD)), which plays a decisive role in epidermal differentiation and specifically labels epidermal cells committed to differentiation 25 . While the number of NICD-positive cells in the Plexin-B1/Plexin-B2-deficient epidermis was not significantly changed at embryonic day 15.5 ( Fig. 2f, g and Supplementary Fig. 2f)-the developmental stage when stem cell hyperproliferation was most pronounced (Fig. 1f, g) -Notch signaling became markedly increased at embryonic day 16.5 ( Fig. 2f, h). This increase was due to elevated numbers of NICD-positive cells in suprabasal layers of the epidermis ( Supplementary Fig. 2g). Consistently, we found an expansion of K10-positive differentiating cells in suprabasal layers at later embryonic stages, i.e. at E18.5 (Fig. 2i, j and Supplementary Fig. 2h, i). Concomitantly, the number of K14-positive stem cells in the Plexin-B1/Plexin-B2-deficient epidermis normalized at E18.5 (Fig. 2i, k). As no differences in apoptosis rate were detected ( Supplementary Fig. 2j), we conclude that hyperproliferation of Plexin-B1/Plexin-B2-deficient epidermal stem cells coincides with overcrowding and cell shape anisotropy in the basal layer, which is followed-with a temporal delay-by increased differentiation to restore tissue size and architecture.
Plexin-B1/Plexin-B2 inhibit YAP activity in response to mechanical forces. We next sought to identify the downstream signaling mechanism by which Plexin-B1/Plexin-B2 balance cell division rates to cell density. The transcriptional regulator YAP is known as a key factor in sensing cell density and shape to regulate cell proliferation 7,26 , and has been shown to play critical functional roles in skin development, homeostasis, expansion, repair, and cancer [27][28][29][30] . In the epidermis, high cell densities suppress YAP activity and stem cell divisions 30,31 . We therefore examined how stem cell overcrowding and shape anisotropy in the Plexin-B1/Plexin-B2-deficient embryonic epidermis impact on YAP signaling. Surprisingly, we observed that both the levels of active, dephosphorylated YAP (Fig. 3a-c) as well as the mRNA expression level of the YAP target gene ctgf ( Supplementary Fig. 3a-c) were elevated in embryonic epidermal stem cells lacking Plexin-B1 and Plexin-B2. This suggested that Plexin-B1/Plexin-B2-deficient epidermal stem cells fail to restrict YAP activity in response to crowding. To test this hypothesis, we cultured primary mouse keratinocytes at different densities and analyzed YAP localization as well as mRNA expression levels of YAP target genes. At low cell densities, when YAP is predominantly localized to the nucleus and active, these parameters were uninfluenced by the loss of Plexin-B1/Plexin-B2 ( Supplementary Fig. 3d, e). At high cell densities, however, Plexin-B1/Plexin-B2-deficiency impeded shuttling of YAP out of the nucleus (Supplementary Fig. 3h) Fig. 3i). Interestingly, the suppressive effect of Plexin-B1/ Plexin-B2 on YAP activity at high cell density was dependent on the presence of calcium in the culture medium ( Supplementary  Fig. 3f-i), which induces the formation of cadherin-based cell-cell adhesion complexes 32 . To further assess the role of Plexin-B1/ Plexin-B2 in mechanosensation of tissue stress anisotropy caused by crowding, we seeded primary mouse keratinocytes on circular or square micropatterned surfaces with identical surface areas, on which cells experience isotropic (circles) and anisotropic (squares) traction stresses 18 . Fully in line with a function of Plexin-B1/ Plexin-B2 in mechanosensation of crowding, the dependence of YAP localization on Plexin-B1/Plexin-B2 was more pronounced under anisotropic than under isotropic traction stress conditions (Fig. 3d, e). Finally, to directly test for a requirement of Plexin-B1/ Plexin-B2 in mechanosensation of crowding-induced mechanical forces, we subjected primary mouse keratinocyte monolayers to static stretch (for 12 h) to expand the cell-substrate adhesive surface area, followed by release of tension resulting in a transient increase in monolayer compression 18 (Fig. 3f). While in control cells compression triggered a reduction in nuclear YAP, Plexin-B1/Plexin-B2-deficient cells failed to respond (Fig. 3g, h).
To test whether the loss of B-plexin-mediated suppression of YAP activity in response to mechanical forces was causative for tissue overgrowth in vivo, we pharmacologically inhibited YAP in embryos. To do so, the YAP inhibitor verteporfin 33,34 was injected into pregnant female mice at E15.5, and embryonic epidermal thickness and proliferation were analyzed 24 h later at E16.5. Indeed, verteporfin treatment largely normalized both epidermal thickness (Fig. 3i, j) and epidermal stem cell proliferation rates (Fig. 3k, l) of Plexin-B1/Plexin-B2-deficient embryos as compared to control embryos. Taken together, these data indicate that Plexin-B1/Plexin-B2 are required for mechanosensation in epidermal stem cells, and suppress YAP activity in response to mechanical forces generated by crowding-induced lateral compression. Moreover, they demonstrate causality of the B-plexin-mediated suppression of YAP activity for the limitation of epidermal tissue growth.
Plexin-B1/Plexin-B2 mediate mechanosensation through stabilization of adhesive cell-cell junctions. We next aimed to elucidate the precise molecular mechanism through which Plexin-B1/Plexin-B2 limit mechanosensitive YAP activity and epidermal stem cell proliferation. Plexin-B1 and Plexin-B2 are known to serve as receptors for class-4 transmembrane semaphorins, and to mediate forward signaling in response to ligand binding 13 . In addition, both Plexin-B1 as well as Plexin-B2 can act as ligands and induce reverse signaling via binding to class-4 transmembrane semaphorins 35,36 . First, we examined expression levels of Plexin-B1/Plexin-B2 ligands, i.e. of semaphorin 4A (Sema4A), semaphorin 4C (Sema4C), and semaphorin 4D (Sema4D), in primary mouse keratinocytes and found these to be largely unaltered by Plexin-B1/Plexin-B2 deficiency (Supplementary Fig. 4a-c). To investigate the effect of an activation of semaphorin-plexin signaling on YAP activity, we treated primary mouse keratinocytes with (1) recombinant Sema4C, a ligand for Plexin-B2 13 , (2) recombinant Sema4A or Sema4D, ligands for both Plexin-B1 and Plexin-B2 13 , or (3) recombinant Plexin-B1 or Plexin-B2, and measured the mRNA expression levels of the YAP target genes ctgf and cyr61. However, neither activation of semaphorin-plexin forward nor of reverse signaling was sufficient to suppress YAP target gene expression ( Supplementary Fig. 4d, e). Interestingly, in Xenopus, Plexin-A1 has been described to interact in trans with Plexin-A1 on other cells, and this homophilic binding has been shown to depend on the presence of calcium ions 37 . We found that the localization of Plexin-B1 and Plexin-B2 to cell-cell contacts of primary mouse keratinocytes was stabilized in the presence of calcium ( Fig. 4a and Supplementary Fig. 4f), which was intriguing given the strong calciumdependency of YAP activity control by Plexin-B1/Plexin-B2 (Supplementary Fig. 3d-i). Structure-function experiments in a renal tubular epithelial cell line showed that this localization of Plexin-B2 to cell-cell contacts relied on its extracellular domain and was independent of its intracellular domain ( Supplementary  Fig. 4g). To test whether Plexin-B1 and Plexin-B2 mediate homophilic binding in the presence of calcium, we measured the adhesion of primary mouse keratinocytes to recombinant Plexin-B1 and Plexin-B2. Indeed, while control cells strongly adhered to recombinant Plexin-B1 and Plexin-B2, Plexin-B1/Plexin-B2deficient cells adhered less efficiently (Fig. 4b). In contrast, we did not detect any binding of primary mouse keratinocytes to recombinant Sema4C ( Supplementary Fig. 4h). Interestingly, recombinant Sema4A interfered with adhesion of primary mouse keratinocytes to recombinant Plexin-B1 ( Supplementary Fig. 4i), suggesting that Sema4A could compete with homophilic plexin interactions. Taken together, these data show that Plexin-B1/ Plexin-B2 localize to cell junctions and promote cell-cell adhesion via a homophilic-binding mechanism.
Plexin-B1/Plexin-B2 regulate dynamic crowding-induced changes of cortical stiffness. Cadherin-based cell junctions not only connect, via α-catenin, to the cortical actomyosin network, but also contribute to its biogenesis 39 . The cortical actomyosin network, in turn, generates cortical tension and promotes cadherin clustering and stability at cell-cell contact sites 39,40,43,44 . We therefore investigated whether Plexin-B1/Plexin-B2 impact on the organization and regulation of the actomyosin network. Indeed, cortical F-actin intensity at contact sites of dividing epidermal stem cells and their respective neighbor cells was less pronounced in the epidermis of Plexin-B1/Plexin-B2-deficient embryos as compared to control embryos (Fig. 5a, b). Moreover, the levels of phosphorylated myosin light chain 2 (pMLC-2), a marker for actomyosin contractility, were markedly reduced ( Fig. 5c, d). In order to probe for a potential requirement of Plexin-B1/Plexin-B2 in the regulation of the actomyosin network by mechanical forces, we exposed control and Plexin-B1/Plexin-B2-knockout keratinocytes to static stretch and abrupt compression. These analyses revealed that, in contrast to control cells, Plexin-B1/Plexin-B2-deficient cells were unable to modulate the levels of pMLC-2 in response to crowding (Fig. 5e, f). We then asked whether this impaired modulation of pMLC-2 levels would correlate with an impaired regulation of cell surface area and cortical stiffness in response to crowding. When released from stretch, control primary mouse keratinocytes rapidly reduced their surface area (Fig. 5g). In striking contrast, keratinocytes lacking Plexin-B1/Plexin-B2 failed to respond by a surface area decrease (Fig. 5g). To examine the crowding-induced regulation of cortical stiffness, we performed atomic force microscopy (AFM)-mediated force indentation spectroscopy on micropatterns at different cell densities. While control cells, as expected, showed a linear inverse correlation of cortical elastic modulus and cell density, Plexin-B1/Plexin-B2-deficient cells exhibited no correlation (Fig. 5h). Taken together, these data demonstrate that Plexin-B1/Plexin-B2 are required for dynamic modulation of the actomyosin network, cell surface area, and cortical stiffness in response to lateral compression.
A number of studies have revealed that remodeling of the actomyosin network is required for mitotic cell rounding [45][46][47][48] . Moreover, B-plexins have been reported to induce cell rounding in COS-7 49 and in HeLa cells 50 . Indeed, we observed an impaired rounding of Plexin-B1/Plexin-B2-deficient epidermal stem cells in mitosis ( Fig. 5i-k). In addition to regulating mitotic cell rounding, the cortical actomyosin network has also been shown to control mitotic spindle orientation, which plays a critical role in epidermal stem cell biology 45,47,48,[51][52][53] . Moreover, plexins have been identified as critical determinants of mitotic spindle orientation in neuroepithelial progenitors and renal tubular epithelial cells 23,54 . In line with this, we found mitotic spindle orientation of embryonic epidermal stem cells to be perturbed by loss of Plexin-B1/Plexin-B2, with Plexin-B1/Plexin-B2-deficient stem cells dividing at lower angles, i.e. more parallel to the basement membrane than cells of control embryos (Fig. 5l, m). Taken together, these data further corroborate the function of Plexin-B1/Plexin-B2 in modulation of actomyosin-dependent processes in epidermal stem cells.
Plexin-B1/Plexin-B2 control YAP activity and cell proliferation in basal cell carcinoma. Next, we asked whether Plexin-B1/ Plexin-B2-mediated mechanosensation of proliferation-induced tissue crowding could also be relevant in skin disease. Consistent with the low rate of stem cell divisions in the adult murine epidermis (Fig. 1a, b) 19,20 , genetic inactivation of Plexin-B1 and Plexin-B2 at adult stages using a tamoxifen-inducible K14- Fig. 3 Plexin-B1/Plexin-B2 inhibit YAP activity in response to mechanical forces. a Confocal images of murine epidermis at E15.5 and E16.5 immunostained for active YAP (red) and K14 (green). Scale bar, 25 µm. b, c Quantification of the data in a (percentage of basal cells) (mean ± s.d.; E15.5: n = 5 mice per genotype, E16.5: n = 4 mice per genotype; b p = 0.0047, c p = 0.0078; two-sided unpaired t-test). d, e Primary mouse keratinocytes on d circular or e square micropatterns immunostained for Yap (green) after 3 h of 1.8 mM Ca 2+ . Representative images are shown on the left, quantifications of Yap localization are shown on the right (mean ± s.d.; circles: control: n = 73 micropatterns from 5 mice, PlexDKO: n = 73 micropatterns from four mice; N > C: p = 0.000008, N = C: p = 0.012038, N < C: p = 0.000008; squares: control: n = 69 micropatterns from five mice, PlexDKO: n = 69 micropatterns from four mice; N > C: p < 0.000001, N = C: p < 0.000001, N < C: p = 0.000050; two-sided unpaired t-test). Blue: DAPI. Scale bars, 25 μm. f Schematic illustration of abrupt crowding experiments. Confluent monolayers were exposed to 120% static uniaxial stretch, which was abruptly released. Cells were analyzed at the indicated time points (R5': 5 min after release; R15': 15 min after release). g, h Representative YAP immunofluorescence images g and quantification of nuclear YAP h upon crowding (mean ± s.d.; n = 5 independent experiments with >200 cells/condition/experiment; *p = 0.0358, ***p = 0.0004, one-way ANOVA, Tukey test; values are normalized to control 120% stretch; statistics have been done from the non-normalized values; scale bars 20 µm). i-l Pregnant females were injected at E15.5 with vehicle control (DMSO) or verteporfin (i.p., 100 μg/g body weight). Embryos were harvested at E16. 5 To assess the potential pathophysiological relevance of mechanosensation through Plexin-B1/Plexin-B2 in disease, we next analyzed the role of Plexin-B1/Plexin-B2 in skin tumors. To do so, we employed two genetic mouse models for basal cell carcinoma, the most common type of skin cancer 55 . In these mouse models, epidermal stem cell proliferation and tumor formation is driven by tamoxifen-inducible epidermis-specific inactivation of the tumor suppressors Gαs or Patched1, the most frequently mutated gene in human basal cell carcinoma 56 (Fig. 6a and Supplementary Fig. 8a). Interestingly, in both genetic models, mice lacking Plexin-B1 and Plexin-B2 displayed a higher incidence ( Fig. 6b and Supplementary Fig. 8b) and larger sizes of tumors (Fig. 6c, d and Supplementary Fig. 8c, d) than the respective control mice. Furthermore, disruption of Plexin-B1 and Plexin-B2 resulted in a strong increase in cell proliferation (Fig. 6e, f and Supplementary Fig. 8e, f) and a marked upregulation of YAP activity (Fig. 6g, h and Supplementary Fig. 8g, h). In summary, these data indicate that Plexin-B1/Plexin-B2 limit YAP activity and proliferative capacity not only during embryonic development but also in skin cancer in mice.
To investigate a potential role of Plexin-B1 and Plexin-B2 for basal cell carcinoma progression in humans, we next studied primary human basal cell carcinoma cells. Interestingly, in these cells, Plexin-B2 protein expression was almost lost and Plexin-B1 protein expression was markedly lower than in benign human primary keratinocytes (Fig. 7a), supporting the concept that loss of Plexin-B1/Plexin-B2 could provide a mechanistic basis for basal cell carcinoma progression also in humans. The strongly reduced expression levels of Plexin-B1 and Plexin-B2 correlated with an impaired ability of the primary human cancer cells to exclude YAP from the nucleus at high cell densities ( Supplementary Fig. 9a, b). To test for a functional role of Bplexins in the control of YAP activity in human primary basal cell carcinoma cells, we engineered cells to re-express Plexin-B2 ( Supplementary Fig. 9c). Indeed, Plexin-B2-expressing primary human basal cell carcinoma cells displayed less nuclear YAP and lower mRNA expression levels of YAP target genes than GFPexpressing control cells (Fig. 7b-d). In summary, these data suggest that the B-plexin-dependent mechanochemical feedback could also be relevant in basal cell carcinomas in human patients.

Discussion
Cell divisions within the jammed basal layer of the mouse embryonic epidermis result in crowding and mechanical stress anisotropy. Our study is in line with a concept, in which crowding acts as a central checkpoint in the control of epidermal morphogenesis by triggering mechanisms to reinstate stem cell density. These mechanisms include differentiation and extrusion of differentiating cells from the stem cell layer by upward efflux 18,57-59 , as well as suppression of stem cell divisions by mechanical feedback 41 . Our data now identify the transmembrane receptors Plexin-B1 and Plexin-B2 as key to this feedback by coupling mechanosensation of cell density to the biochemical control of YAP activity and cell proliferation. Our work demonstrates that the Plexin-B1/Plexin-B2-mediated mechanoresponse to crowding is particularly relevant for proper morphogenesis of the early embryonic epidermis where proliferation rates are high. The sharp decrease of Plexin-B1 and Plexin-B2 expression levels at late embryonic stages and, in particular, the absence of epidermal abnormalities after induction of Plexin-B1/Plexin-B2 deficiency in adult mice indicate that-in contrast to early development-the mechanosensation of crowding does not coordinate epidermal homeostasis when proliferation rates are low. This is consistent with previous studies showing that in the adult interfollicular epidermis, stem cells divide only upon demand to replace delaminating cells, and thus crowding does not occur 10,60 . Intriguingly, we found that mechanosensation through Plexin-B1/Plexin-B2 regains biological relevance in the adult murine epidermis for the suppression of cancer cell proliferation. In line with this, we observed that expression levels of Plexin-B1 and Plexin-B2 in primary human basal cell carcinoma cells were low, and that reexpression of Plexin-B2 was sufficient to suppress YAP activity. Taken together, these data strongly suggest that loss of Plexin-B1/ Plexin-B2 could represent a mechanistic basis for the loss of contact inhibition, a hallmark of cancer that has remained poorly understood on a molecular level 61,62 . In support of this concept, Plexin-B1 has been shown to also suppress proliferation of human renal adenocarcinoma 63 and melanoma cell lines 64 , and to inhibit primary tumor growth of human melanoma cells in a xenograft mouse model 64 . Moreover, in human patients, Plexin-B1 is downregulated in renal cell carcinoma 63 and melanoma 65 , and low expression levels of Plexin-B1 correlate with bad prognosis in particular subsets of breast cancer [66][67][68] . It has to be noted, however, that the functions of plexins in cancer are highly contextdependent, and that in other settings, plexins can also promote tumor progression 13,14,69,70 . On a molecular level, this functional Fig. 4 Plexin-B1/Plexin-B2 mediate mechanosensation through stabilization of adhesive cell-cell junctions. a Primary mouse keratinocytes were cultured with 70 µM Ca 2+ . 1.8 mM Ca 2+ was added ("high calcium"). Shown are confocal images of immunostainings for Plexin-B1 (green) and Plexin-B2 (red). Scale bar, 25 µm. b, f Specific adhesion of primary mouse keratinocytes to the recombinant extracellular portions of b Plexin-B1 or Plexin-B2, and f Ecadherin (mean ± s.d.; n = 4 mice per genotype; p = 0.0124 for Plexin-B1, p = 0.0332 for Plexin-B2; p = 0.0006 for E-cadherin; two-sided unpaired t-test). c Live cell imaging of primary mouse keratinocytes expressing E-cadherin-mRuby. Shown are representative epifluorescence still images of cell divisions. Arrows: dividing cells; arrowheads: cell-cell contacts. Time: hours:minutes format. Scale bar, 10 µm. d, h, j Confocal images of the epidermal basal layer at E15.5 immunostained for d E-cadherin (red), h α-catenin (red), and j a-18 (=α-catenin tension-sensitive epitope antibody; green). Cell divisions are contoured by dashed lines. Scale bar, 10 µm. e, i, k Quantification of e E-cadherin, i α-catenin, and k a-18 immunofluorescence intensities at cell-cell contacts between a dividing cell and its immediate neighbors (box plot with minimum, first quartile, median, third quartile and maximum). e control: n = 207 cell-cell contacts of 34 dividing cells from three mice, PlexDKO: n = 209 cell-cell contacts of 34 dividing cells from three mice. i control: n = 228 cell-cell contacts of 37 dividing cells from three mice, PlexDKO: n = 244 cell-cell contacts of 39 dividing cells from three mice. k control: n = 222 cell-cell contacts of 36 dividing cells from three mice, PlexDKO: n = 219 cell-cell contacts of 37 dividing cells from three mice. Two-sided Mann-Whitney U test for e, i, and k: p < 0.0001. g Super-resolution structured illumination microscopy images of the epidermal basal layer at E15.5 immunostained for Plexin-B2 (red) and E-cadherin (green). Scale bar, 2 µm. The dashed boxed area is magnified on the right. Scale bar, 1 µm. l Representative a-18 (magenta) and nuclear (DAPI; cyan) immunofluorescence confocal images of primary mouse keratinocyte monolayers exposed to uniaxial static stretch and subsequent release. Scale bars, 20 µm. versatility of plexins is reflected by their association with multiple co-receptors and signaling molecules that significantly impact on plexin function 71,72 .
The extracellular domain of several plexins has been shown to adopt different conformations ranging from a nearly closed, ringlike form to a more-open, chair-like form 17,73,74 . Recently, a particular plexin, Plexin-D1, has been reported to sense shear stress-induced mechanical forces in endothelial cells 17 . This function in mechanosensation required the opening of the ringlike conformation of the Plexin-D1 extracellular domain, but was independent of the binding of semaphorins 17   B1 and Plexin-B2 by semaphorins is not sufficient to regulate the activity of the mechanotransducer YAP. The similar structures of the extracellular domains of plexins 13,73 suggest that the mechanism of mechanosensation by semaphorin-independent conformational changes could be shared by different plexins including Plexin-B1 and Plexin-B2.
In addition to B-plexins, several other proteins have been described to play a role in mechanosensation and mechanotransduction in the epidermis. These include the mechanosensitive ion channels Piezo1 in keratinocytes 75 and Piezo2 in Merkel cells 76,77 , E-cadherin [38][39][40]78,79 , and integrins 29,80 . Ecadherin molecules organize into microclusters, the formation of which requires homophilic binding at cell-cell contacts 81 . Our data show that B-plexins aggregate into similar clusters, which also depend on homophilic interactions. Albeit spatially separated from E-cadherin aggregates, these B-plexin microclusters are required for proper E-cadherin localization at the plasma membrane of epidermal stem cells.
As opposed to B-plexins, which we have identified as suppressors of YAP activity, integrins promote YAP activity in the epidermis via Src and FAK signaling 29 . Interestingly, Plexin-B1 has been shown to inhibit integrins and FAK in several cell lines in a semaphorin-dependent manner 49,82 . It is therefore conceivable that B-plexins-in addition to their ligand-independent role in mechanosensation-also mediate a semaphorin-induced inhibition of integrin activity in the epidermis, and thereby suppress a functionally antagonistic mechanosensitive pathway.
In summary, our work uncovers a critical requirement for Plexin-B1 and Plexin-B2 in the control of epidermal stem cell proliferation by mechanical forces. Whether the function of Plexin-B1 and Plexin-B2 in mechanosensation is specific for crowding-induced compression remains open to future research; however, it seems more likely that they could respond to a wider spectrum of different mechanical stimuli, a property that is known from other types of mechanosensors such as mechanosensitive ion channels 83 . Given the wide expression of Plexin-B1 and Plexin-B2 in epithelial and non-epithelial cells both during development as well as in the adult 13,84,85 , it is tempting to speculate that mechanosensation by Plexin-B1 and Plexin-B2 could be instrumental in physiology and pathophysiology of multiple tissues.
Uncropped western blots can be found in the Source data file.   were purchased from R&D Systems. A fragment of human Plexin-B1 cDNA (encoding amino acids 1-535) was cloned into the pcDNA5 vector using HindIII (R0104S, NEB) and XhoI (R0146S, NEB) restriction sites with addition of a Cterminal 6xHis tag (KHHHHHH). The expression plasmid was transfected into Expi293F cells (Invitrogen) according to the manufacturer's instructions. The supernatant containing secreted Plexin-B1 (amino acids 20-535)-6xHis was collected after 7 days and purified using a two-step purification protocol developed at Quantitative RT-PCR. Total RNA from murine skin, murine epidermis, and primary mouse keratinocytes was isolated using Direct-zol RNA Microprep Kits (Zymo) according to the manufacturer's instructions. Genomic DNA was removed by on-column DNAse digestion, and cDNA was generated by reverse transcription using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher). qRT-PCR was performed with SYBR Green Supermix (BioRad) in a Real Time Quantitative Thermal Cycler (BioRad). For absolute quantifications, mouse genomic DNA was used as a standard. For relative quantifications, gene expression changes were normalized to gapdh or actb expression. Complete lists of all primers used are supplied as Supplementary Tables 1 and 2.
Cell lines. MDCK cells, a canine renal tubular epithelial cell line, were cultured in MEM supplemented with 10% FBS 23 .
Primary cells. Primary mouse keratinocytes were isolated and cultured essentially as described 78 . Briefly, the skin of newborn mice was harvested using sterile forceps, and incubated in 1 ml dispase (5 mg/ml) in culture medium (CnT-07; Cellntec) in a 2-ml-microcentrifuge tube over night at 4°C. The skin was then transferred to a 10-cm-Petri dish, and washed with PBS. Using forceps, the epidermal sheet was separated from the dermis, transferred with the basal layer facing downwards onto 500 µl of accutase (Cellntec) in a 6-cm-Petri dish, and incubated for 30 min at RT. Keratinocytes were washed out of the epidermal basal layer using 2 ml of culture medium, collected by centrifugation, and seeded onto collagen type 1 (30 µg/ml, Biochrom, cat no. L7213)-coated 10-cm-Petri dishes. Primary mouse keratinocytes were kept in culture medium (CnT-07; Cellntec) at 37°C and 5% CO 2 . Only low passage cells (max. 2 passages) were used for experiments. For the analysis of semaphorin-plexin forward and reverse signaling, primary mouse keratinocytes were incubated with recombinant proteins (25 nM) for 8 h prior to RNA isolation.
Human primary epidermal keratinocytes (hPEKs) were purchased from CellnTec and were grown in keratinocyte growth medium (cat. no. CnT-PR; CellnTec). Human primary basal cell carcinoma cells (hPBCCs) were purchased from Celprogen and were grown in human basal cell carcinoma cell line complete media with serum (cat. no. M77015-08S, Celprogen).
Lentiviral and retroviral transduction. To generate an E-cadherin-mRuby lentiviral construct, the human cDNA of E-cadherin was amplified by PCR and fused to the mRuby2 sequence using overlap/extension PCR. The fused cDNA was ligated into pWPXL via Mlu1 and Spe1 restriction sites. Triple-myc-tagged full-length mouse Plexin-B2 91 was ligated into the pENTR1A entry vector using SalI/NotI restriction enzymes. By recombination using the Gateway LR Clonase (Invitrogen), the cDNA was then inserted into the lentiviral expression vector pINDUCER20 92 , which allows for doxycycline-inducible expression of triple-myc-tagged full-length mouse Plexin-B2. To generate a respective control plasmid, GFP was ligated into the pENTR1A entry vector using NcoI/NotI restriction enzymes, followed by  Fig. 7 B-plexins suppress YAP activity in human basal cell carcinoma cells. a Human primary epidermal keratinocytes (hPEKS) and human primary basal cell carcinoma cells (hPBCCs) were lysed, and proteins were detected using the indicated antibodies. b, c hPBCCs engineered to express GFP ("control") or myc-tagged Plexin-B2 ("Plexin-B2") and cultured at dense conditions were immunostained for YAP (red) and myc (green). Blue: DAPI. Representative confocal images are shown in b with dashed boxed areas depicted in the right panel. A quantification of YAP nuclear intensity is shown in c (mean ± s.d.; n = 3 independent experiments; p = 0.0495; two-sided unpaired t-test). d hPBCCs were engineered to express GFP ("control") or myc-tagged Plexin-B2 ("Plexin-B2"), and cultured at dense conditions. Relative mRNA expression levels of the YAP target genes ctgf and cyr61 were determined by quantitative RT-PCR (mean ± s.d.; n = 5 independent experiments; p = 0.0123 for ctgf, p = 0.0002 for cyr61; two-sided unpaired t-test).
Immunofluorescence staining. For whole-mount immunofluorescence stainings, the epidermis was harvested from the dorsal skin of E15.5 embryos using forceps, fixed in 4% PFA for 30 min, washed twice in 50 mM glycine in PBS, followed by washing twice in PBS. After permeabilization with PBST (0.5% Triton X-100 in PBS) for 20 min and blocking with 4% FCS in PBST for at least 1 h, the epidermis was incubated with primary antibodies (diluted in 2% FCS in PBST) at 4°C overnight. After three times washing in PBST, samples were incubated with secondary antibodies and DAPI for 3 h, and washed. All processing of samples was done in 12-well or 96-well plates. Samples were then mounted on glass slides using Fluoromount (Dako). Images were taken using a confocal laser scanning microscope (Zeiss LSM 700).
For immunofluorescence stainings of paraffin-embedded sections, tissues (dorsal skin of mouse embryos or adult mouse skin/tumor tissue) were fixed in 4% PFA at 4°C overnight, embedded in paraffin according to standard laboratory protocols, sectioned at 5 μm, and mounted on glass slides. Antigen retrieval was done by boiling in a steam pressure pot in 10 mM sodium citrate buffer pH 6.0 for 10 min. Sections were permeabilized with PBST (0.2% Triton X-100 in PBS) for 10 min, blocked with 4% FCS in PBST for at least 1 h and incubated with primary antibodies (diluted in 2% FCS in PBST) at 4°C overnight, washed with PBS, and incubated with secondary antibodies and DAPI at room temperature for 1 h. After three times washing in PBS, sections were mounted with Fluoromount (Dako). Images were taken using a confocal laser scanning microscope (Zeiss LSM 700).
For immunofluorescence staining of cryotome sections, dorsal skin of mouse embryos or adult mouse skin were fixed in 0.2% PFA overnight at 4°C, followed by one day of incubation in 30% sucrose (in PBS). Tissues were embedded in OCT medium, frozen on dry ice, and sectioned using a cryotome (thickness of sections 25 μm), and mounted on glass slides. Sections were postfixed in 4% PFA at 4°C for 30 min, washed twice in 50 mM glycine in PBS, permeabilized with PBST (0.2% Triton X-100 in PBS), blocked with 4% FCS in PBST for at least 1 h and incubated with primary antibodies (diluted in 2% FCS in PBST) overnight at 4°C. Sections were washed three times in PBS, incubated with secondary antibodies and DAPI at room temperature for 1 h. After three times washing in PBS, sections were mounted with Fluoromount (Dako). Images were taken using a confocal laser scanning microscope (Zeiss LSM 700).
For immunofluorescence stainings of primay mouse keratinocytes, keratinocytes (seeded into μ-Slide eight-well (ibidi) or onto micropatterns) were fixed in 4% PFA for 30 min, washed three times with PBS, permeabilized with PBST (0.2% Triton X-100 in PBS) for 10 min, blocked with 4% FCS in PBST for at least 1 h, incubated with primary antibodies (diluted in 2% FCS in PBST) at 4°C overnight, washed with PBS and incubated with secondary antibodies and DAPI at room temperature for 1 h. After three times washing in PBS, cells were mounted with Fluoromount (Dako). Images were taken using a confocal laser scanning microscope (Zeiss LSM 700).
For immunostainings on silicone elastomers, cells were fixed in 4% PFA for 10 min room temperature, permeabilized with 0.3% Triton X-100 in PBS, and blocked in 5% bovine serum albumin (BSA). Samples were subsequently incubated overnight in primary antibody in 1% BSA/0.3% Triton X-100/PBS, followed by washing in PBS and incubation in secondary antibody in 1% BSA/0.3% Triton X-100/PBS. Finally, samples were mounted in Elvanol. Images were collected by laser scanning confocal microscopy (SP8X; Leica) with Leica Application Suite software (LAS X version 2.0.0.14332), using ×40 immersion objectives. Images were acquired at room temperature using sequential scanning of frames of 1 µm-thick confocal planes (pinhole 1) after which 10 planes encompassing complete cell nuclei were projected as a maximum intensity confocal stack. Images were collected with the same settings for all samples within an experiment.
BrdU labeling. Pregnant females were injected i.p. with 200 µl of BrdU (Sigma, cat. no. B5002; 7.5 mg/ml in PBS) 2 h prior to collection of embryos. BrdU incorporation was detected by immunohistochemical staining using an anti-BrdU antibody.
Immunohistochemical staining. For immunohistochemical stainings of paraffinembedded sections, tissues were fixed in 4% PFA at 4°C overnight, embedded in paraffin according to standard laboratory protocols, sectioned at 5 μm, and mounted on glass slides. Antigen retrieval was done by boiling in a steam pressure pot in 10 mM sodium citrate buffer pH 6.0 for 10 min. For immunohistochemical staining of NICD, sections were incubated with BLOXALL (ImmPRESS Excel Staining Kit; Vector laboratories, cat. no. MP-7601) to inactivate endogenous peroxidases for 10 min, washed in PBS, permeabilized with PBST (0.2% Triton X-100 in PBS) for 10 min, blocked with 4% FCS in PBST for at least 1 h, and incubated with anti-NICD antibody (diluted in 2% FCS in PBST) at 4°C overnight, washed with PBS, followed by usage of the ImmPRESS Excel Staining Kit (Vector laboratories, cat. no. MP-7601). Sections were then dehydrated and mounted with Pertex. For immunohistochemical staining of BrdU, sections were incubated in 0.3% H 2 O 2 in methanol for 20 min, washed in PBS, and incubated in 2 M HCl at 37°C for 30 min. Sections were then washed with PBST (0.2% Triton X-100 in PBS), blocked with 4% FCS in PBST for at least 1 h, and incubated with anti-BrdU antibody (diluted in 2% FCS in PBST) at 4°C overnight. After washing with PBST, the ABC HRP Kit (Vector laboratories, cat. no. PK-4001) and the DAB Peroxidase (HRP) Substrate Kit (Vector laboratories, cat. no. SK-4100) were used according to the manufacturer's instructions. Sections were then dehydrated and mounted with Pertex.
TUNEL assay. TUNEL assays were performed on paraffin-embedded sections of the dorsal skin of embryos using the DeadEnd Fluorometric TUNEL System (Promega, cat. no. G3250) according to the manufacturer's instructions.
Immunoprecipitation. For protein immunoprecipitation, primary mouse keratinocytes were lysed in ice-cold buffer containing 50 mM Tris pH 7.6, 150 mM NaCl, 1% NP-40, 1.8 mM CaCl 2 with complete EDTA-free Protease Inhibitor cocktail (Roche). Lysates were cleared by centrifugation, and supernatants were incubated with 2 µg anti-E-cadherin antibody in the presence of Protein G PLUS Agarose beads (Santa Cruz Biotechnology). Agarose beads were washed four times with lysis buffer, and immunocomplexes were eluted in Laemmli buffer. Samples were then subjected to immunoblot analysis.
Surface biotinylation assay. Confluent primary mouse keratinocytes in 10-cm Petri dishes were washed twice with ice-cold PBS followed by incubation with 8 ml of Biotin-NHS (Sigma, cat. no. 203112) in PBS (0.25 mg/ml) for 10 min at 4°C with gentle shaking. Afterwards, the reaction was quenched with the addition of 6 ml Tris-HCl (50 mM, pH 7,4). Cells were scraped off the dish, collected in a 50 ml tube, pelleted by centrifugation at 4°C, and washed three times with ice-cold PBS. The pellet was resuspended in 800 µl of RIPA buffer (50 mM Tris-HCl, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% sodium deoxycholate; 0.1% SDS, 5 mM MgCl 2 ; pH 7.4) containing cOmplete Protease Inhibitor Cocktail (Roche, cat. no. 4693132001), and sonicated three times. After centrifugation at 4°C, supernatants were incubated with Pierce Streptavidin Agarose (ThermoFisher, cat. no. 20353) for 90 min at 4°C. Agarose beads were washed four times with ice-cold lysis buffer, and boiled in Laemmli buffer before analysis on SDS-PAGE followed by a Western blotting.
Live cell imaging. Live cell imaging of primary mouse keratinocytes was performed using a Thunder Imaging System (Leica) at 37°C in a CO 2 humidified incubation chamber in cell culture medium (CnT-07, Cellntec).
Adhesion assay. Adhesion assays were performed essentially as described 37 . Mixed Cellulose Ester 96-well plates (Millipore, cat. no. MSHAS4510) were coated without or with recombinant proteins (5 µg/ml) at 4°C overnight. Wells were then blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature. After washing with PBS, 1 × 10 4 primary mouse kerationocytes (in culture medium with 1.8 mM calcium) were added per well, and allowed to adhere for 30 min at 37°C and 5% CO 2 . Afterwards, non-adherent cells were removed by washing twice with PBS. For some control wells, non-adherent cells were not removed by washing and all seeded cells remained in the well. The CellTiter-Glo Luminescent Cell Viability Assay (Promega, cat. no. G7572) was used according to the manufacturer's protocol. The following three conditions were analyzed: (condition 1) Primary keratinocytes were added to uncoated wells. Wells were not washed and nonadherent cells were not removed, i.e. all seeded cells remained in the well; (condition 2) Primary keratinocytes were added to uncoated wells. Wells were washed and non-adherent cells were removed; (condition 3) Primary keratinocytes were added to wells coated with recombinant proteins. Wells were washed and nonadherent cells were removed; specific adhesion of primary keratinocytes (in %) was then calculated by the following formula: specific adhesion = (RLU condition 3 −RLU condition 2)/RLU condition 1 = (RLU specifically adhered cells minus RLU non-specifically adhered cells) divided by RLU total cells = RLU of coated well after removal of non adherent cells À RLU of non coated well after removal of non adherent cells RLU of non coated well without removal of non adherent cells Pharmacological treatments. Mice at the age 6 weeks were injected with tamoxifen (1 mg; i.p.) for 5 consecutive days. One week after the start of tamoxifen injections, the dorsal hair was shaved. Two days later, DMBA (25 µg = 97.5 nmol, dissolved in acetone) was applied to the shaved back skin. Starting 2 weeks after the treatment with DMBA, 12-O-Tetradecanoylphorbol-13-acetate (TPA; 4 µg = 6.5 nmol, dissolved in acetone) was applied to the same site twice per week for a total of 14 weeks. The analyzed genotypes were "contr." (genotype plxnb1 flox/flox ;plxnb2 flox/flox ) and "PlexDKO ind" (genotype K14-CreERT;plxnb1 flox/flox ;plxnb2 flox/flox ). Mice were regularly monitored for the appearance of skin lesions, and did not develop papillomas.
Analysis of mitotic spindle angle. Paraffin-embedded sections of the murine epidermis at E16.5 were immunostained for phospho-histone H3 and stained with DAPI, and confocal images were acquired. The mitotic spindle angle, defined as the angle between the plane of the basal cell layer and the plane of mitosis (axis transecting two daughter nuclei) was quantified using ImageJ by an observer blinded to mouse genotypes.
Quantitative image analysis. Segmentation analyses were done based on Ecadherin immunofluorescence stainings using the Tissue Analyzer plug-in in ImageJ. Cell area and shape (major axis/minor axis; roundness: 4×[area]/(π × [major axis] 2 ) were quantified by using the "area" and "shape description" functions of ImageJ. The number of cells per area (cell density) was quantified manually.
To quantify mean fluorescence intensities, the segmented line tool of Fiji (line width 5 pixels) was used.
All quantifications were done by an observer blinded to genotypes. For crowding experiments, images were analyzed using Fiji 93 . To quantify nuclear YAP, fields were randomly selected based exclusively on the presence of nuclei, as assessed by DAPI staining. Areas of interest were generated using automated thresholding of the DAPI staining, after which mean fluorescence intensities of nuclear YAP intensity was quantified within the areas of interest from maximum projection images obtained as described above. The measured mean nuclear intensities were normalized to DAPI intensity of the corresponding individual nuclei to account for possible unevenness in sample topology. To measure pMLC-2 intensity, pMLC-2 signal intensity at junctions was measured by automatic thresholding followed by subtraction of cytoplasmic background signal. Cell area was measured by manually tracing α18 fluorescence. Cell orientation was quantified by measuring the orientation angle (the angle between the long axis and a line parallel to the x-axis of the image which was aligned to the direction of stretch) of the major Feret diameter of areas of interest corresponding to traced α-18-positive adherens junctions.
Micropatterns. Micropatterned adhesive surfaces were generated using the PRIMO optical module (Alvéole, France) controlled by the Leonardo plugin (V3.3, Alveole) mounted on a Nikon TI-E inverted microscope (Nikon Instruments) equipped with a Super Plan FLuor ×20 ELWD lens (Nikon) lens and a DMD-based UV (375 nm). To generate circular and square micropatterns, both 100 µm diameter, were projected onto plasma-cleaned (Corona Treater, ETP), PLLgPEGpassivated (0.1 mg/ml PLL-g-PEG (PLL (20)-g [3.5]-PEG (2), SuSoS) 35 mm glassbottom dishes (Ibidi). Patterned areas were then washed multiple times with PBS and conjugated with a uniform coating of 10 µg/ml fibronectin and 38.75 µg/ml collagen for 1 h at +37°C. The substrates were then washed with PBS, prior to seeding 100 K of mouse keratinocytes onto each 35 mm dish. Keratinocyte monolayers were allowed to proliferate on the patterns for 24 h, at which time they were subjected to AFM or fixed and processed for immunofluorescence and quantification analyses. For the analysis of YAP localization (Fig. 3d, e), only micropatterns with similar cell densities were compared.
Crowding experiments. The custom-built uniaxial cell stretcher has been described in detail previously 42,94 . Polydimethylsiloxane (PDMS) elastomers were prepared from a two-component formulation (Sylgard 184, Dow Corning) by mixing base and crosslinker in a ratio of 40-1 (weight/weight) to obtain substrates with 50 kPa stiffness. Chambers were UV sterilized for 1 h and finally coated with fibronectin (20 µg/ml) in phosphate-buffered saline (PBS) for 1 h at 37°C prior to cell seeding. 350,000 cells per elastomer (4 cm 2 ) were seeded 16 h before experiment start. Before initiation of experiments, culture medium was replaced by medium containing 200 µM Ca 2+ to promote cell-cell contact formation. Cells were then exposed to 20% static stretch for 12 h and fixed with 4% PFA either directly when under stretch or 5 or 15 min post release. For each independent experiment, cells from independent mice were used.
Atomic force microscopy. AFM measurements were performed on micropatterns generated on glass bottom dishes (35 mm Ibidi). Measurements were done using a JPK NanoWizard 2 (Bruker Nano) atomic force microscope mounted on an Olympus IX73 inverted fluorescent microscope (Olympus) and operated via JPK SPM Control Software v.5. Spherical silicon dioxide beads with a diameter of 3.5 μm glued onto tipless silicon nitride cantilevers (NanoAndMore, CP-PNPL-SiO-B-5) with a nominal spring constant of 0.08 N m −1 were used. For all indentation experiments, forces of up to 4 nN were applied, and the velocities of cantilever approach and retraction were kept constant at 2 μm s −1 ensuring detection of elastic properties only. All analyses (>50 cells per experiment/condition) were performed with JPK Data Processing Software (Bruker Nano). Prior to fitting the Hertz model to obtain cell Young's Modulus (Poisson's ratio of 0.5), the offset was removed from the baseline, contact point was identified, and cantilever bending was subtracted from all force curves.
All experiments presented in the manuscript were repeated in at least three independent experiments/biological replicates.