Supplementary Figure 3: ECM mechanical cues regulate lipid synthesis by controlling SREBP1/2 activation. | Nature Cell Biology

Supplementary Figure 3: ECM mechanical cues regulate lipid synthesis by controlling SREBP1/2 activation.

From: Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP

Supplementary Figure 3

a, A simplified scheme illustrating how SREBP is regulated by sterols (see text). Left: sterols retain SCAP/SREBP at the ER by binding SCAP and Insig1 proteins; a minor portion of SCAP/SREBP complexes continuously shuttles through the Golgi apparatus and is transported back to the ER3,4. Right: absence of sterols induces SCAP/SREBP accumulation at the Golgi apparatus where SREBP are cleaved by Site-1 Protease (S1P), releasing the transcriptionally-active nuclear form of SREBP. b, LDLR-luciferase in MDA231 cells transfected with the indicated siRNAs in combination with expression plasmids encoding for mouse full-length SREBP1 or SREBP2, whose cDNA is insensitive to siSREBP mixes A and B. Mean expression in the control was set to 1, and all other samples are relative to this; data are mean and single points; n≥4 independent biological samples per condition; unpaired Mann-Whitney tests. c, Accumulation of cholesterol (Filipin) and neutral lipids (ORO) in MCF10ATk1 cells treated with YM for 24 hours is inhibited by knockdown of SREBP1/2. Quantifications and n in Supplementary Table 2. Scale bars 5 μm. d, Co-localization of endogenous SREBP2 with an ER marker (KDEL-mCherry, false colors) in control cells (DMSO), or with a Golgi marker (GM130) upon 2 hours of treatment with YM in MCF10ATk1 cells. Scale bars 10 μm. At least 30 cells per condition. e, Quantification of the ER, Golgi and nuclear localization of endogenous SREBP2 during the time-course YM treatment shown in Fig. 3d. Data are mean with range; n = 5 independent slides per condition; unpaired Mann-Whitney tests for Golgi localization. f, Representative immunofluorescence for endogenous SREBP2 in 3T3L1 cells treated with YM, blebbistatin or plated on soft ECM hydrogels for 6 hours. At least 50 cells per condition. Scale bars 10 μm. g, Representative immunofluorescence for endogenous SREBP2 in RPE1 cells treated with YM or plated on soft ECM hydrogels for 6 hours. At least 50 cells per condition. Scale bars 10 μm. h, Western blotting for endogenous mature SREBP2 in RPE1 cells treated with YM or FM. i, Quantification of SREBP2 nuclear localization in the presence of vehicle (EtOH), cycloheximide (CHX) and CHX + YM shown in Fig. 3f. n = 5 independent slides per condition; data are mean and single points; unpaired Mann-Whitney test. j, Luciferase assay with a highly unstable luciferase mRNA/protein reporter (CMV-luc2P_ARE) in MCF10ATk1 cells. Cells were treated for 3 hours with vehicle (EtOH) or 100 μg/ml cycloheximide (CHX) before harvesting. Mean expression in EtOH was set to 1; n = 6 independent biological samples per condition; data are mean and single points; unpaired Mann-Whitney test. The images in panels b, d, f, g and h are representative of at least two independent experiments with similar results. All n values are pooled across independent experiments. Source data in Supplementary Table 3.

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