Extended Data Fig. 8: Nuclear compression regulates mitochondrial mechanotransduction.
a, MCF10A-RAS were cultured for 24 h on soft hydrogels and then subjected to 6 μm vertical confinement (COMPRESS) for 3 h (n = 16 cells from one representative experiment; compression was evident upon visual inspection in all experiments). b, Representative pictures of YAP localization in MCF10A-RAS cells subjected to 6 μm vertical confinement. Scale bar 10 μm. Refers to Fig. 8g. c, Immunostainings for NESPRIN2 confirming defective assembly of the LINC complexes in Sun1-Sun2 KO MEFs. Scale bar 10 μm. d, YAP localization in Sun1-Sun2 KO MEFs (n = 195 (wt) and n = 179 (Sun1/2 KO) from three experiments). Scale bar 10 μm. e, YAP localization in MEFs transfected with increasing doses of dominant-negative GFP-NESPRIN1-KASH or GFP-NESPRIN2-KASH (n = 42 (EMPTY), n = 34 (NES1-KASH low level), n = 35 (NES1-KASH high level), n = 40 (NES2-KASH low level), n = 29 (NES2-KASH high level) from two experiments). Scale bar 10 μm. f, YAP localization in MCF10A-RAS cells transfected as in j (n = 56 (EMPTY), n = 64 (NES1-KASH low level), n = 45 (NES1-KASH high level), n = 43 (NES2-KASH low level), n = 55 (NES2-KASH high level) from two experiments). Scale bar 10 μm. g, Representative pictures of mitochondrial length in MCF10A-RAS cells subjected to 6 μm vertical confinement. Scale bar 10μm. Refers to Fig. 8h. h, Representative pictures of YAP localization in MCF10A-RAS cells subjected to 6 μm vertical confinement. Scale bar 10 μm. Refers to Fig. 8i. i, Mitochondrial length analysis in MCF10A-RAS cells pre-treated for 1 h with inhibitors of ROCK-MLCK (YM) and then subjected to 6 μm vertical confinement (COMPRESS) for 2 h with inhibitors. (n = 53 (DMSO), n = 70 (YM), n = 65 (YM COMPRESS), n = 56 (siMFN1a YM COMPRESS), n = 47 (siMFN1b YM COMPRESS) and n = 49 (MIEF1SA YM COMPRESS) from two experiments). j,k, YAP localization (j) and mitochondrial length analysis (k) and in MEFs cells cultured on soft hydrogel for 24 h and then subjected to 6 μm vertical confinement (COMPRESS) for 2 h. (j n = 43 (STIFF), n = 41 (SOFT), n = 39 (SOFT COMPRESS) and n = 25 (SOFT MIEF1SA COMPRESS); k n = 29 (STIFF), n = 31 (SOFT), n = 43 (SOFT COMPRESS), n = 33 (SOFT MIEF1SA COMPRESS) from two experiments). Data are presented as mean values ± s.d. (d-f, i-k) or mean and single points (a). p-values were calculated based on two-tailed Student’s t-tests. In d this was calculated on nuclear YAP. Source numerical data are available in the source data.
