Supplementary Figure 2: YAP/TAZ activity is inconsequential for lipid synthesis and SREBP activation in response to inhibition of ROCK/MLCK. | Nature Cell Biology

Supplementary Figure 2: YAP/TAZ activity is inconsequential for lipid synthesis and SREBP activation in response to inhibition of ROCK/MLCK.

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

Supplementary Figure 2

a, Staining for cholesterol in MCF10ATk1 depleted of YAP/TAZ. Scale bar: 10 μm. Quantifications and n in Supplementary Table 2. Scale bar 10 μm. b, Staining for cholesterol in MCF10ATk1 stably infected with pBABE TAZ 4SA or empty pBABE control, and treated with YM for 24 hours. Slightly enhanced lipids in TAZ 4SA cells is in line with Ref. 2. Quantifications and n in Supplementary Table 2. Scale bar: 10 μm. c, 8XGTIIC-luciferase reporter assay for YAP/TAZ activity in MDA231 cells transfected with control siRNA (siCO.), YAP/TAZ siRNA (siYT), cotransfected with the YAP/TAZ inhibitor NF2/Merlin expression plasmid, or treated with YM. Mean expression in the control was set to 1, and all other samples are relative to this; n = 4 independent biological samples pooled across 2 independent experiments for each bar; unpaired Mann-Whitney tests. d,e,f, qPCR in MCF10ATk1 (d) MDA231 (e) and 3T3L1 (f) cells treated for the indicated times with YM, FM or DMSO. Data are relative to GAPDH levels; mean expression in control cells was set to 1, and all other samples are expressed relative to this; n ≥ 4 independent biological samples pooled across 2 independent experiments for each bar; multiple unpaired two-tailed Student’s t-tests. g,h, LDLR-luciferase reporter assay for SREBP activity in MDA231 cells transfected in g with control siRNA (siCO.), YAP/TAZ siRNA (siYT) or SREBP1/2 siRNAs (siSREBP A and B), in h with a NF2/Merlin expression plasmid. Mean expression in the control was set to 1, and all other samples are relative to this; n≥4 independent biological samples pooled across at least 2 independent experiments for each bar; unpaired Mann-Whitney tests. i, Uptake of the fluorescent Bodipy-C11 fatty acid was measured by flow cytometry in MCF10ATK1 cells treated with DMSO (D) or YM for 24 hours. n=8 independent biological samples pooled across 3 independent experiments; unpaired two-tailed Student’s t-test. Increased fatty acid uptake is also evident from accumulation of lipid species containing linoleic acid, an essential fatty acid provided by the serum (see Ref. {Romani:2018hf}). j, MCF10ATK1 cells were treated with DMSO or YM for 24 hours with 13C2-acetate in the culture medium and subjected to mass-spectrometry analysis of total fatty acids. M+8 and M+10 indicate fatty acids isotopologs containing 8 or 10 13carbon atoms respectively, which derive from acetate through acetyl-CoA and fatty acid synthesis. Mean isotopolog levels in DMSO-treated cells were set to 1, and levels in YM-treated cells are expressed relative to this; n = 5 independent biological samples per condition in one single experiment; unpaired two-tailed Student’s t-tests. The images in panels a and b are representative of at least two independent experiments with similar results. Data are mean and single points. Source data in Supplementary Table 3.

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