Extended Data Fig. 3: DRP1 is required for the mechano-regulation of YAP-TAZ.
a, Expression levels (CPM, counts per million) of selected proliferation and YAP-TAZ target genes from RNA sequencing of D2.0R cells, selected among the genes upregulated on a soft ECM upon DRP1 shRNA (n = 3 in a single experiment). b, YAP localization in MCF10A-RAS cells treated with ROCK-MLCK inhibitors (YM) and Drpitor1a for 3 h (n = 91 cells (DMSO), n = 105 (YM) and n = 123 (YM DRP1A) from three experiments). Scale bar 10 μm. c, YAP localization in MCF10A-RAS cells treated with the SMIFH2 inhibitor of formin actin-nucleating proteins and with Drpitor1a for 3 h (n = 71 cells (DMSO), n = 78 (SMIFH2) and n = 81 (SMIFH2 DRP1A) from three experiments). Scale bar 10 μm. d, Immunoblotting of extracts from wt and Dnm1l KO MEFs (Drp1 KO). Consistent results were obtained in n = 3 experiments. e, YAP localization in 3T3L1 cells cultured in sparse (LD) and dense (HD) conditions, treated with Drpitor1a. (n = 60 (LD), n = 610 (HD) and n = 625 (HD DRP1A) from three experiments). f, YAP localization in HaCaT cells cultured at high density and either subjected to uniaxial stretching or treated with Drpitor1a for 3 h (n = 543 cells (HD), n = 621 (HD STRETCH) and n = 352 (HD DRP1A) from two experiments). Scale bar 20 μm. g, Immunoblotting of extracts from wt and NF2 KO MCF10A. Consistent results were obtained in n = 3 experiments. h, YAP localization in MCF10A cells (WT) treated with ROCK-MLCK inhibitors and in NF2-knockout MCF10A cells (NF2 KO) transiently transfected to reconstitute the expression of Nf2. Where indicated, cells were treated with Drpitor1a (n = 120 cells (wt DMSO), n = 114 (wt YM), n = 121 (wt YM DRP1A), n = 83 (NF2 KO), n = 51 (NF2 KO Nf2) and n = 72 (NF2 KO Nf2 DRP1A) from three experiments). i, EdU incorporation assay in 3T3L1 cells cultured on soft hydrogels and treated with Drpitor1a for 24 h (n = 134 cells (STIFF), n = 95 (SOFT) and n = 103 (SOFT DRP1A) from three experiments). j, EdU incorporation assay in 3T3L1 cells cultured at high density and treated with Drpitor1a for 24 h (n = 81 cells (LD), n = 343 (HD) and n = 391 (HD DRP1A) from three experiments). k,l, Cell area was measured in MCF10A-RAS cells cultured on a soft ECM for 24 h (k) or at high density for 48 h (l) and treated with Drpitor1a (k, n = 60 (STIFF), n = 60 (SOFT), n = 60 (SOFT DRP1A), n = 60 (SOFT MDIVI) from two experiments; l, n = 60 (LD), n = 60 (HD), n = 60 (HD DRP1A) from two experiments). m,n, SREBP2 (m) and YAP localization (n) in MCF10A-RAS cells treated with inhibitors of ROCK-MLCK (YM) or cultured on a soft ECM. Where indicated, cells were also treated with antioxidant compounds (NAC, TROLOX or MITOTEMPO) for 3 h (m, n = 93 cells (DMSO), n = 82 (YM), n = 56 (YM TROLOX), n = 65 (YM NAC), n = 87 (YM MITO), n = 56 (STIFF), n = 42 (SOFT),n = 62 (SOFT NAC), n = 53 (SOFT TROLOX), n = 41 (SOFT MITO); n, n = 78 (DMSO), n = 65 (YM), n = 41 (YM NAC), n = 67 (YM TROLOX), n = 73 (YM MITO), n = 55 (STIFF), n = 42 (SOFT), n = 39 (SOFT NAC), n = 43 (SOFT TROLOX), n = 39 (SOFT MITO) from two experiments). Scale bar 10 μm. Data are presented as mean values and single points (a,i,j), mean ± s.d. (b,c,e,f,h,m,n) or violin plots with median and quartiles (k,l). p-values were calculated based on two-tailed Student’s t-tests. In b,c,e,h, p-values were calculated on nuclear YAP levels. Source numerical data and unprocessed blots are available in the source data and Supplementary Table 1.
