Extended Data Fig. 6: ECM stiffness regulates mitochondrial fission through DRP1.
a, Quantification of mitochondria length in D2.0R cells transfected with mitoRFP and treated with YM (n = 128 (DMSO) n = 117(YM) mitochondria pooled across 10 cells per condition in two independent experiments for each bar; unpaired two-tailed Student’s t-tests). The same set of images was analysed by automated classification of mitochondrial length (centre) and by MFC analysis (right). Data are mean and single mitochondria (left), mean and s.d. (centre) and mean and single pictures (right). b, Quantification of mitochondria content by MitoTracker staining in MCF10A-RAS and D2.0R cells treated with YM. Median level in the control was set to 1, and other samples are relative to this (n = 7 biologically independent samples pooled across three independent experiments for MCF10A-RAS; n = 4 biologically independent samples pooled across two independent experiments for D2.0R). c, Quantification of mitochondrial DNA content in MCF10A-RAS cells treated with YM, based on qPCR for three different mtDNA loci. mtDNA levels are relative to genomic DNA (gDNA) levels; mean level in the control was set to 1, and other samples are relative to this (n = 6 biologically independent samples from two independent experiments). d, qPCR for DRP1 in MCF10A cells treated with YM. mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 6 biologically independent samples from three independent experiments). e, Immunoblotting for endogenous total and phosphorylated DRP1 (p616, p637) from extracts of MCF10A-RAS cells treated with ROCK/MLCK inhibitors. Equal total proteins were loaded in each lane, and GAPDH was used as loading control. f,g. qPCR to control for efficient knockdown of DRP1 in MCF10A-RAS (f) or in D2.0R (g). mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 4 biologically independent samples from two independent experiments for each bar; unpaired two-tailed Student’s t-test in MCF10A-RAS, Dunnet’s test in D2.0R). h. Immunoblotting for endogenous DRP1 from extracts of MCF10A-RAS cells transfected with the indicated siRNAs. Equal total proteins were loaded in each lane, and GAPDH was used as loading control. i. Quantification of mtROS in MCF10A-RAS cells with knockdown of the indicated fission factors, and treated with YM. Median intensity in the control were set to 1, and other samples are relative to these (n = 8 biologically independent samples pooled across four independent experiments for siCO bars; n = 4 biologically independent samples pooled across two independent experiments for other bars; unpaired two-tailed Student’s t-tests). j-m. qPCR in MCF10A-RAS to control for efficient knockdown of the FIS1, MFF, MIEF1 and MIEF2 fission factors. mRNA expression data are relative to GAPDH levels; mean level in the control was set to 1, and other samples are relative to this (n = 4 biologically independent samples from two independent experiments for each bar; Dunnet’s tests). n. Oxygen Consumption Rate (OCR) analysis performed on monolayers of MCF10A-RAS cells transfected with control (siCO) or DRP1 siRNA and plated on stiff or soft Matrigel substrata (n = 20 biologically independent samples pooled across two independent experiments). o. OCR analysis on cells were treated with Drpitor1a (n = 20 biologically independent samples pooled across two independent experiments). p. OCR analysis performed on monolayers of D2.0R cells stably expressing control (shCo.) or DRP1a shRNA and cultured on stiff or soft Matrigel substrata (n = 20 biologically independent samples pooled across two independent experiments). q. OCR analysis on cells were treated with Drpitor1a (n = 20 biologically independent samples pooled across two independent experiments). Images in e,h are representative of two independent experiments with similar results. Unprocessed blots in Source Data Extended Data Fig. 6. Data are mean and single points, except n-q (mean and s.d. - shaded areas). See Source Data Extended Data Fig. 6.
