Fig. 2: DRP1 is required for the mechanoregulation of SREBP1/2 and lipogenesis.
a, MCF10A-RAS cells were treated with ROCK–MLCK inhibitors (YM) for 3 h. DRP1 was inhibited with Drpitor1a (DRP1A), Mitochondrial division inhibitor 1 (MDIVI1) or siRNA transfection. The percentages of cells displaying SREBP2 staining at the endoplasmic reticulum (ER) or in the nucleus (Nuc) were quantified (n = 95 (DMSO), 63 (YM), 87 (YM DRP1A), 88 (YM MDIVI1) and 75 cells (YM siDRP1) from three experiments). Scale bar, 10 μm. b, SREBP2 localization in MCF10A-RAS cells cultured on fibronectin hydrogels for 24 h (n = 80 (stiff), 64 (soft), 73 (soft DRP1A), 68 (soft MDIVI1) and 99 cells (soft siDRP1) from three experiments). Scale bar, 10 μm. c, MCF10A-RAS cells expressing Myc-tagged SCAP were treated as in b and the percentages of cells displaying SCAP immunofluorescence staining at the endoplasmic reticulum or concentrated at the Golgi apparatus were quantified (n = 40 (stiff), 51 (soft), 41 (soft DRP1A) and 43 cells (soft MDIVI1) from three experiments). Scale bar, 5 μm. d, SREBP2 localization in D2.0R cells with inhibition of ROCK–MLCK (YM) or DRP1 (DRP1A or shRNAa/b) (n = 101 (DMSO), 124 (YM), 106 (YM DRP1A), 98 (YM shDrp1a) and 102 cells (YM shDrp1b) from three experiments). Scale bar, 10 μm. e,f, SREBP2 localization in WT and Dnm1l KO (Drp1 KO) MEFs treated with YM (e) or cultured on fibronectin hydrogels for 24 h (f) (n = 103 (DMSO), 105 (YM), 92 (YM DRP1A), 69 (Drp1 KO) and 76 cells (Drp1 KO YM) in e; n = 56 (WT stiff), 71 (WT soft), 54 (KO stiff) and 61 cells (KO soft) in f; both from three experiments). Scale bars, 10 μm. g, SREBP2 localization in 3T3L1 cells cultured on large or small micropatterned islands for 24 h (n = 49 (large), 51 (small) and 63 cells (small DRP1A) from three experiments). Scale bar, 5 μm. h, Quantitative PCR (qPCR) in MCF10A-RAS cells cultured on stiff (E = ~50 kPa) or soft (E = ~0.2 kPa) collagen I-coated hydrogels for 24 h and treated with DRP1 inhibitors. The messenger RNA (mRNA) levels are relative to GAPDH levels and normalized to the stiff controls (n = 6 samples from three experiments). i,j, Quantification of lipid droplets by Oil Red O (ORO) staining (i) and of cholesterol by ultraviolet-fluorescent Filipin (j) in MCF10A-RAS cells treated with YM for 24 h and with DRP1 inhibitors (n = 50 cells in i and n = 53 cells in j; both from three experiments). Scale bars, 10 μm. The data are presented as means ± s.d. (a–g) or means and single points (h–j). Statistical significance was determined by two-tailed Student’s t-test. In a, b and d–g, this was calculated on nuclear SREBP2. Source numerical data are available.
