Fig. 8: MIEF1 phosphorylation mediates MIME. | Nature Cell Biology

Fig. 8: MIEF1 phosphorylation mediates MIME.

From: RETRACTED ARTICLE: Mitochondrial mechanotransduction through MIEF1 coordinates the nuclear response to forces

Fig. 8

a, SREBP2 localization in MCF10A-RAS cells depleted of MIEF1/2 and transfected to reconstitute the WT, SE and SA MIEF1 variants. Where indicated, cells were treated with ROCK–MLCK inhibitors (YM) (n = 62 (siCO DMSO), 71 (siCO YM), 51 (siMIEF1/2a DMSO), 56 (siMIEF1/2a YM), 58 (siMIEF1/2a MIEF1 WT low level YM), 59 (siMIEF1/2a MIEF1 WT medium level YM), 60 (siMIEF1/2a MIEF1 WT high level YM), 58 (siMIEF1/2a MIEF1 SA low level YM), 66 (siMIEF1/2a MIEF1 SA medium level YM), 68 (siMIEF1/2a MIEF1 SA high level YM), 69 (siMIEF1/2a MIEF1 SE low level YM), 63 (siMIEF1/2a MIEF1 SE medium level YM) and 70 cells (siMIEF1/2a MIEF1 SE high level YM) from three experiments). b, YAP localization in MCF10A-RAS cells reconstituted as in a (n = 91 (siCO DMSO), 102 (siCO YM), 87 (siMIEF1/2a DMSO), 99 (siMIEF1/2a YM), 104 (siMIEF1/2a MIEF1 WT low level YM), 89 (siMIEF1/2a MIEF1 WT medium level YM), 91 (siMIEF1/2a MIEF1 WT high level YM), 88 (siMIEF1/2a MIEF1 SA low level YM), 98 (siMIEF1/2a MIEF1 SA medium level YM), 101 (siMIEF1/2a MIEF1 SA high level YM), 81 (siMIEF1/2a MIEF1 SE low level YM), 73 (siMIEF1/2a MIEF1 SE medium level YM) and 75 cells (siMIEF1/2a MIEF1 SE high level YM) from three experiments). c, SREBP2 localization in MCF10A-RAS cells with overexpression of WT and SA MIEF1 variants on stiff glass (n = 66 (untrasfected), 59 (WT low level), 58 (WT high level), 61 (SA low level) and 69 cells (SA high level) from three experiments). d, YAP localization in MCF10A-RAS cells as in c (n = 104 (untrasfected), 63 (WT low level), 64 (WT high level), 65 (SA low level) and 67 cells (SA high level) from three experiments). e, qPCR in MCF10A-RAS cells stably expressing MIEF1 variants, transfected with MIEF1/2 siRNA and cultured on collagen I-coated hydrogels for 24 h. The heat maps indicate the mRNA levels of NRF2 targets (HMOX1 and NQO1), SREBP1/2 targets (DHCR7 and FASN) and YAP–TAZ targets (CYR61 and ANKRD1) in single samples relative to GAPDH levels and normalized to the mean control (n = 6 from three experiments). ind, doxycycline induction. f, Uptake of FITC-labelled cystine in MCF10A-RAS cells (n = 10 (siCO DMSO), 10 (siCO YM), 10 (siMIEF1/2a DMSO), 10 (siMIEF1/2a YM), 10 (siMIEF1/2a MIEF1 WT DMSO), 10 (siMIEF1/2a MIEF1 WT YM), 10 (siMIEF1/2a MIEF1 SA DMSO), 6 (siMIEF1/2a MIEF1 SA YM), 6 (siMIEF1/2a MIEF1 SE DMSO) and 6 replicates (siMIEF1/2a MIEF1 SE YM) from three experiments). g,h, YAP localization (g) and mitochondrial length analysis (h) in MCF10A-RAS cells cultured for 24 h on soft hydrogels and then subjected to 6 μm vertical confinement (compress) for 3 h (n = 70 (stiff), 84 (soft) and 78 (soft compress) in g; n = 52 (stiff), 68 (soft) and 71 (soft compress) in h; both from three experiments). i, YAP localization in MCF10A-RAS cells pre-treated for 1 h with inhibitors of ROCK–MLCK (YM) and then subjected to 6 μm vertical confinement (compress) with inhibitors for 2 h (n = 36 (DMSO), 41 (YM), 40 (YM compress), 39 (siMFN1a YM compress), 40 (siMFN1b YM compress) and 27 (MIEF1 SA YM compress) in i; n = 53 (DMSO), 70 (YM), 65 (YM compress), 56 (siMFN1a YM compress), 47 (siMFN1b YM compress) and 49 (MIEF1 SA YM compress) in j; both from two experiments). j, Adipogenic differentiation (FABP4 immunostaining) of 3T3L1 cells on micropatterned islands, treated with the Drpitor1a DRP1 inhibitor or expressing the indicated shRNAs (n = 69 (large), 79 (small), 61 (small DRP1A), 59 (small shDrp1a), 68 (small shDrp1b), 59 (small shMief1/2a) and 66 cells (small shMief1/2b) from three different experiments). Scale bars, 10 μm. k,l, Adipogenic differentiation of 3T3L1 cells at high density (HD), treated with the Drpitor1a DRP1 inhibitor or expressing the indicated shRNAs and scored by AdipoQ expression levels (k) or LipidTOX staining (l). The mRNA levels in k are relative to Gapdh levels and normalized to the low-density controls (n = 6 from three experiments). Scale bars in l, 10 μm. m, Adipogenic differentiation of 3T3L1 cells on micropatterned islands expressing the indicated shRNAs (Nrf2 and Scap) or the constitutively active TAZ4SA (n = 69 (large), 79 (small), 56 (small shNrf2a), 49 (small shNrf2b), 63 (small shScapa), 67 (small shScapb) and 57 cells (small TAZ4SA) from three different experiments). n, Adipogenic differentiation of 3T3L1 cells at high density (HD) expressing the indicated shRNAs or the constitutively active TAZ4SA. AdipoQ mRNA levels are relative to Gapdh levels and normalized to the low-density controls (n = 6 from three experiments). o, Adipogenic differentiation of 3T3L1 cells transfected with the WT and SA MIEF1 variants and cultured under low-density conditions without spatial confinement (n = 59 (LD), 67 (LD MIEF1 WT), 69 (LD MIEF1 SA) and 498 (HD) from three experiments). Scale bars, 5 μm. The data are presented as means ± s.d. (a–d and g–i) or means and single points (f and j–o). Statistical significance was determined by two-tailed Student’s t-test. This was calculated on nuclear YAP (b, d, g and i) or punctate mitochondria (h). P values for e and all source numerical data are available as source data.

Source data

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