Supplementary Figure 5: ROCK/MLCK inhibition induces phenotypes different from NPC1 inhibition and similar to Lipin-1 inhibition.
From: Extracellular matrix mechanical cues regulate lipid metabolism through Lipin-1 and SREBP
a, Filipin staining in MCF10ATk1 cells treated with DMSO, YM or with the U18666A NPC1 inhibitor (3 μM) for the indicated times. NPC1 inhibition blocks the transport of extracellular LDL-cholesterol to the ER, causing lysosomal cholesterol accumulation. At least 30 cells per condition. Scale bar 10 μm. g, MCF10ATk1 cells were treated as in f. Arrowheads indicate cholesterol accumulation (blue) in structures encircled by the lysosomal membrane marker LAMP2 (red). At least 30 cells per condition. Scale bar 10 μm. h, immunofluorescence for OSBP-mCherry transfected in MCF10ATk1 cells. OSBP is a cytosolic protein that accumulates to the Golgi apparatus when the levels of cholesterol in the ER are low; U18666A and serum deprivation (NO FCS) serve as positive controls for decreased ER cholesterol levels. On the right: cells were scored based on differential localization of OSBP At least 24 cells were scored per condition. Scale bar 10 μm. d, A simplified scheme illustrating how SREBP is regulated by Lipin-1 and ARF1 (see text). e, qPCR validation of the effects of COPI siRNAs. Data are relative to GAPDH levels; mean expression in controls 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. f, Phenotypic appearance of the Golgi apparatus in MCF10ATk1 cells treated for 24 hours with YM, as assayed with the Golgin97 or Giantin markers. DAPI (blue) serves as nuclear counterstain. At least 50 cells per condition. Scale bar 10 μm. g, Phenotypic appearance of Golgi membranes (GFP-Rab6) in RPE1 cells treated for 24 hours with YM or with 100 μM Propranolol. At least 30 cells per condition. Scale bar 10 μm. h, Localization of transfected KDEL-mCherry in GFP-Rab6 RPE1 cells treated for 6 hours with YM or with Propranolol. At least 30 cells per condition. The images in panels a, b, c, f, g, and h are representative of at least two independent experiments with similar results. n values are pooled across independent experiments. Source data in Supplementary Table 3.
