Journal: Nature Communications
Article Title: Sterol regulatory element binding protein 1 couples mechanical cues and lipid metabolism
Figure Lengend Snippet: RhoA and acto-myosin regulate the activity of hSREBP1 and dSREBP. a Screening of low density lipoprotein receptor promoter-luciferase activity in MDA-MB-231 cells transfected with constructs expressing firefly (LDLR-Luc) and renilla (Rluc) luciferase and either control siRNA (siCTL) or siRNAs targeting genes encoding geranylgeranyl pyrophosphate transferase I (GGTase1) protein substrates. b Western blot analysis of MDA-MB-231 and Mahlavu cells 48 h after transfection with siCTL or RhoA targeting siRNAs (siR#1 and siR#2). Hsp90 was used as loading control. mSREBP indicates mature protein. c Western blot analyses of immunoprecipitated (IP) RhoA in MCF-10A cells treated DMSO (as control), 1 μM cerivastatin (STAT), 1 μM cerivastatin and 20 μM GGPP (STAT+GGPP), 5 μM GGTI-298 or 5 μM FTI-277. IgGs were used as IP control. d LDLR-Luc assay in MCF-10A cells 12 h after transfection with pcDNA3-GFP control plasmid, pcDNA3-GFP-RhoA G14V construct with a 6 h DMSO treatment, pcDNA3-GFP-RhoA G14V construct with a 6 h Latrunculin A (G14V + Lat.A) treatment, or pcDNA3-GFP-RhoA T19N construct. e LDLR-Luc assay in MCF-10A cells treated with either DMSO as control or C3 for 24 h. f LDLR-Luc assay in MCF-10A cells treated with either DMSO as control, Y-27632, or Blebbistatin (Blebbist.) for 24 h. g BODIPY 493/503 staining of lipid droplets (in red) in Mahlavu cells treated with either DMSO, C3 or Y-27632 for 24 h. Scale bar, 15 μm. h , i BODIPY 493/503 staining of lipid droplets (in red) and immunofluorescence analysis of dSREBP (in green) and phosphorylated myosin light chain (pMLC2, in magenta) in Drosophila larval fat body from h flies expressing either Luciferase or dRhoA RNAi , or dRhoA RNAi and treated with fatostatin (FTS) and i wild-type flies treated with either DMSO or Y-27632. Scale bar, 20 μm. Graphs bars in ( c – f ) represent mean ± s.d. of n = 3 biological replicates. Values in ( d – f ) are expressed as Relative Luminometer Units (RLU). Nuclei in ( h – i ) were stained with HOECHST (in blue). Blots and images are representative of n = 3 biological replicates. P value: * P
Article Snippet: Forty-eight hours after transfection of the reporter (i.e. 72 h after siRNA transfection), the cells were lysed in 1 × Glo Lysis Buffer (Promega); Firefly and Renilla luciferase activities were measured using the Dual-Glo Luciferase Assay System (Promega), according to the manufacturer’s instructions, using an Envision Multimode Plate Reader (PerkinElmer).
Techniques: Activity Assay, Luciferase, Multiple Displacement Amplification, Transfection, Construct, Expressing, Western Blot, Immunoprecipitation, Plasmid Preparation, Staining, Immunofluorescence