Extended Data Fig. 1: Forces induce mitochondrial elongation.
a, qPCR in MCF10A-RAS cells transfected with PIEZO1-2 siRNA mixes. The mRNA levels are relative to the GAPDH levels, and normalized to control siRNAs (siCO) (n = 4 samples from two experiments). b-g, Quantification of mitochondrial morphology by mitochondrial fragmentation count (MFC) analysis in MCF10A-RAS cells cultured on hydrogels (b) or treated with YM (c), in MLECs with inducible Talin1 KO (d), in MCF10A-RAS cells cultured in sparse or dense conditions (e), in 3T3L1 cells cultured on micropatterns (f) and in HUVECs subjected to flows (g). The same set of pictures quantified in Fig. 1 were analyzed. h, MCF10A-RAS cells expressing a single-fluorophore excitation-ratiometric ROCK kinase Activity Reporter (ROCKAR-WT) or a version of the same reporter bearing a threonine to alanine mutation of the phosphorylation site (ROCKAR-TA) were transfected with ROCK1/2 siRNA. A lower ratio between the fluorescence emitted in response to excitation at 488 and the fluorescence emitted in response to excitation at 405 nm (Excitation ratio) indicates decreased phosphorylation of the reporter. (n = 59 (siCO), n = 59 (siROCK1/2a), n = 59 (siROCK1/2b) from two experiments). i, qPCR in MCF10A-RAS cells transfected with siRNA mixes targeting ROCK1 and ROCK2. The mRNA levels are relative to the GAPDH levels, and normalized to control siRNAs (siCO) (n = 4 from two experiments). j, MCF10A-RAS cells expressing the ROCKAR sensor were treated with ROCK inhibitors for 3 h (n = 30 (DMSO), n = 30 (Y27632) and n = 30 (FASUDIL) from two experiments). k, Excitation ratio of the ROCKAR sensors in MCF10A-RAS cells cultured on soft and stiff hydrogels for 24 h and treated with Drp1 inhibitor (DRP1A) (n = 30 (STIFF), n = 30 (SOFT), n = 30 (SOFT DRP1A) from two experiments). l, Excitation ratio of the ROCKAR sensors in 3T3L1 cells cultured in small and large micropatterned islands for 24 h (n = 25 (LARGE), n = 25 (SMALL), n = 25 (SMALL DRP1A) from two experiments). m, Excitation ratio of the ROCKAR sensors in MCF10A-RAS cells cultured in sparse and dense conditions for 48 h (n = 50 (LD), n = 50 (HD), n = 50 (HD DRP1A) from two experiments). n, Excitation ratio of the ROCKAR sensors in HaCaT cells cultured in sparse conditions, in dense conditions, and dense then subjected to tonic uniaxial stretching for 3 h (n = 30 (LD), n = 30 (HD), n = 30 (HD STRETCH) from two experiments). o, Excitation ratio of the ROCKAR sensors in MCF10A-RAS cells treated with SMIFH2 or with the non-muscle myosin type II inhibitor for 24 h (n = 30 (DMSO), n = 30 (SMIFH2), n = 30 (BLEBBI) from two experiments). Data are presented as mean values and single points (a,h-o) or box plots with median (centre), quartiles (25% and 75%) and extreme values (whiskers) (b-g). In b,c,e-g, p-values were calculated based on two-tailed Student’s t-tests. Source numerical data are available in the source data.
