Supplementary Figure 1: Lipid accumulation upon ROCK/MLCK inhibition is a general response.

a, Immunofluorescence for F-actin bundles with Phalloidin in MCF10ATk1 cells treated for 6 hours with DMSO or with 20 μM Y27632 and 20 μM ML7 (YM). At least 50 cells per condition. Scale bar: 10 μm. b, qPCR of the YAP/TAZ target CTGF in MCF10ATk1 cells treated as in a for 24 hours. Data are relative to GAPDH levels. Mean expression in controls was set to 1, and all other samples are relative to this. n = 4 per condition, pooled across 2 independent experiments. c, TUNEL assay on MCF10ATk1 cells treated for 24 hours with YM. n = 4 independent biological samples per condition, pooled across 2 independent experiments. d, EdU incorporation assay on MCF10ATk1 cells treated 24 hours with YM. n = 4 independent biological samples per condition. pooled across 2 independent experiments. e, Heatmap of lipid molecules displaying significant accumulation by global metabolomics (mean fold>2.5 P<0.05) in MCF10ATk1 cells treated 6 or 24 hours with YM (n=6 biologically independent samples), as compared to an equivalent dose of DMSO (D, n = 4 biologically independent samples). Each column represents an independent biological replicate; each line represents a single metabolite. AcC acylcarnitines; GP phospholipids; LysoGP lyso-phospholipids; MG monoacylglycerols; DG diacylglycerols; SL sphingolipid metabolism; ST sterols. Triglycerides were not part of this analysis. f-h, lyso-phosphatidylcholine (lyso-PC, f) and ceramide (Cer, g and h) levels in MCF10ATk1 cells treated 24 hours with YM or DMSO, measured by targeted lipidomics. Only the five most abundant and lyso-PC are shown. n=5 biologically independent samples per condition. i, Cholesterol (Filipin) and neutral lipids (ORO) accumulation in MCF10ATk1 mammary epithelial treated with YM at lower concentrations for 24 hours. j, Close-up of a representative YM-treated MCF10ATk1 cell transfected with the lipid-droplet marker Perilipin3-RFP and stained with Bodipy493/503 neutral lipid stain. k, Treatment of cells with YM together with the pan-caspase inhibitor Z-VAD-FMK (ZVAD, 20 μM) excludes activation of SREBP by caspases¹. l, Time-course of lipid accumulation upon YM treatment. m, Cholesterol accumulation in multiple cell lines treated with YM for 24 hours: RPE1 human immortalized retinal pigment epithelium; MDA231 human metastatic breast cancer; 3T3L1 mouse primary fibroblasts; MCF10A human immortalized mammary epithelium; HEK293 human immortalized embryonic kidney; WI38 human adult primary fibroblasts. n, Neutral lipid accumulation in multiple cells treated as in m. Scale bars 5 μm. The images in panels a, i-n are representative of at least two independent experiments with similar results. Data are mean and SD or mean and single points; unpaired two-tailed Student’s t-tests. Source data and quantification of images are providedin Supplementary Table 3.