Fig. 5 | Nature Communications

Fig. 5

From: LINC complex-Lis1 interplay controls MT1-MMP matrix digest-on-demand response for confined tumor cell migration

Fig. 5

Lis1 and LINC complex function is required for invadopodia assembly and activity. a Effect of Lis1 KD on invasion speed of MDA-MB-231 cells expressing H2BGFP in 3D collagen gels polymerized at 37 °C or 20 °C (as in Fig. 1e). n, number of cells analyzed from three independent experiments. Data were transformed using the log transformation y= log(y) to make data conform to normality and analyzed using one-way ANOVA test. b Pericellular collagenolysis (mean Col1-3⁄4C signal per cell normalized to mean intensity of siNT-treated cells ± SEM) by MDA-MB-231 and HT-1080 cells silenced for Lis1 or Nesprin-2 in collagen polymerized at 37 °C (as in Fig. 1k). n, number of cells analyzed from three independent experiments. Kruskal–Wallis test as compared to siNT. c Angular distribution of MT1-MMPmCh endosomes in Lis1-depleted cells in 37 °C or 20 °C polymerized collagen gel (as in Fig. 2b). See Supplementary Movie 6. d Maximal projection of a series of sixteen confocal sections (7.5 μm width) of MDA-MB-231 cells expressing Tks5GFP (green) in 3D collagen gel polymerized at 37 °C (Cyan) stained for cleaved collagen (red) and nucleus (blue). Scale bars, 10 μm. Bottom row, two-by-two channel combinations corresponding to boxed region #2. Scale bar=5 μm. e MDA-MB-231 cells silenced for Lis1 or nesprin-2 embedded in 3D collagen as in d, stained for Tks5GFP (green) and cleaved collagen (red). Right row shows intensity profiles of Tks5GFP, Col1-3⁄4C and DAPI maximum intensities along the long-cell axis (dotted line-enclosed regions). f MDA-MB-231 cells silenced for Lis1 were incubated on a thick fibrous type I collagen layer (cyan). Invadopodia in association with collagen fibrils are labeled for cortactin (green) and Tks5 (red). Insets, two-by-two channel combinations of boxed regions. Tks5 is excluded from cortactin-positive lamellipodia (inset #2). Scale bars=10 μm. g Quantification of Tks5 signal in MDA-MB-231 and HT-1080 cells treated with indicated siRNAs plated on a thick layer of type I collagen. Y-axis indicates ratio of Tks5 area to total cell area normalized to mean value in siNT-treated cells (as percentage) ± SEM. n, number of cells analyzed from three independent experiments. Kruskal–Wallis test. h Quantification of Tks5 signal in MDA-MB-231 expressing GFPKASHext or GFPDN-KASH as in panel g from two independent experiments; Mann–Whitney test. *P < 0.05; ***P < 0.001; ****P < 0.0001

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