Extended Data Fig. 4: General workings of mitochondrial mechanotransduction.
a-d, Mitochondrial length analysis (a and c) and YAP localization (b and d) in MCF10A-RAS cells treated with inhibitors of ROCK-MLCK (YM; a and b) or cultured on soft hydrogels (c and d) and treated with the Drpitor1a inhibitor (DRP1A). (a n = 26 cells (YM), n = 21 (YM DRP1A 0.2microM), n = 22 (YM DRP1A 2microM) and n = 23 (YM DRP1A 10microM); b n = 63 cells (YM), n = 81 (YM DRP1A 0.2microM), n = 73 (YM DRP1A 2microM) and n = 53 (YM DRP1A 10microM); c n = 20 (SOFT), n = 20 (SOFT DRP1A 0.2microM), n = 20 (SOFT DRP1A 2microM) and n = 20 (SOFT DRP1A 10microM); d n = 56 cells (SOFT), n = 57 (SOFT DRP1A 0.2microM), n = 56 (SOFT DRP1A 2microM) and n = 56 (SOFT DRP1A 10microM) from two experiments). e, PCR for mitochondrial mtDNA loci in “deep” rho-zero (ρ0) MCF10A-RAS cells. The expression levels were normalized to nuclear Actin DNA (n = 4 from two experiments). f, Mitochondrial length analysis in MCF10A-RAS cells treated with the FCCP uncoupler of oxidative phosphorylation (n = 78 (DMSO) and n = 80 (FCCP) from three experiments). Scale bar 2 μm. g, Mitochondrial length analysis in MCF10A-RAS cells treated with Ionomycin (i n = 25 (DMSO) and n = 25 (IONO) from two experiments). Scale bar 2 μm. h, Mitochondrial length analysis in wt and Drp1 KO MEFs expressing the ActuAtor system45. Cells were left untreated or treated for 2 h with Rapamycin to activate the ActuAtor system (+Act) and induce fission (n = 26 cells (wt), n = 27 (wt Act), n = 26 (KO) and n = 26 (KO Act) from two experiments). Scale bar 2 μm. i, qPCR of MCF10A-RAS cells with MFN1 knockdown. The mRNA levels are relative to the GAPDH levels, and normalized to the stiff controls (n = 4 from two experiments). j, Mitochondrial length analysis in MCF10A-RAS cells with MFN1 knockdown. Where indicated, cells were treated with Drpitor1a (n = 41 cells (siCO), n = 56 (siMFN1a), n = 51 (siMFN1a DRP1A), n = 48 (siMFN1b), n = 49 (siMFN1b DRP1A) from three experiments). Scale bar, 2 μm. k, Immunoblotting of wt and Mfn1 KO MEFs. Consistent results were obtained in n = 2 experiments. l, YAP localization in Mfn1 KO MEFs (n = 55 cells (wt) and n = 65 (KO) from two experiments). m, Quantification of DRP1 mitochondrial puncta in MCF10A-RAS cells depleted of MFN1 and treated with Drpitor1a (n = 42 cells from two experiments). Scale bar 2 μm. n, Quantification of DRP1 mitochondrial puncta in Mfn1 KO MEFs (n = 43 from two experiments). o, qPCR of MCF10A-RAS cells with PLD6 knockdown. The mRNA levels are relative to the GAPDH levels, and normalized to the stiff controls (n = 4 from two experiments). p, Mitochondrial length analysis in MCF10A-RAS cells with PLD6 knockdown (n = 66 cells (siCO), n = 76 (siPLD6a) and n = 65 (siPLD6b) from two experiments). Data are presented as mean values ± s.d. (a-d,f-h,j,l,p) or mean and single points (e,i,m-o). p-values were calculated based on two-tailed Student’s t-tests. In f,j, this was calculated on punctate mitochondria. Source numerical data and unprocessed blots are available in the source data.
