Extended Data Figure 7: Nfat activity in Mfn2−/− and Prdm16−/− MEFs.
From: Mitofusin 2 maintains haematopoietic stem cells with extensive lymphoid potential
a, Mfn2fl/fl (blue) or Mfn2fl/fl-Vav-Cre (Mfn2−/−) (red) HSCs were stained with DCFDA to visualize intracellular reactive oxygen species levels. Cells were treated with oligomycin for 15 min as a positive control. b, HSC apoptosis levels were analysed by surface staining of Annexin V. c, Intracellular staining for the endoplasmic reticulum stress response chaperone GRP78 in Mfn2fl/fl or Mfn2fl/fl-Vav-Cre (Mfn2−/−) HSCs (red colours) MPPs (green colours) and CMPs (blue colours). Histograms; representative of three biological replicates. d, Calcium flux trace (left) and baseline intracellular Ca2+ (right) in WT LSK cells transduced with IRES–GFP or Mfn2–IRES–GFP lentiviral vectors (e) and with WT or Mfn2−/− MEFs stained with Indo1. Bars, mean ± s.e.m.; n = 3 biological replicates; *P < 0.05; two-tailed Student’s t-test. f, Nfat1 staining (left, scale bars, 5 μm) and fraction of nuclear Nfat1 (right, *P < 0.05) in HSCs transduced with IRES–GFP or Mfn2–IRES–GFP lentiviral vector. Bars, mean ± s.e.m.; n ≥ 10 from two biological replicates; *P < 0.05; two-tailed Student’s t-test. g, Nfat1 staining (left; scale bars, 20 μm) and fraction of nuclear Nfat1 (right; *P < 0.05) in WT or Mfn2−/− MEFs. Bars, mean ± s.d.; n ≥ 20 fields from three independent experiments; *P < 0.05; two-tailed Student’s t-test. h, Nfat luciferase gene reporter activity in WT or Mfn2−/− MEFs. Bars, mean ± s.d.; n = 3 experiments; *P < 0.05; two-tailed Student’s t-test. i, Mitochondrial morphology after transfection of dominant negative Drp1 into HeLa cells (scale bars, 5 μm). Note extreme elongation of mitochondria (arrow), confirming that this vector is functional. j, Nfat1 staining (left, scale bars, 5 μm) and fraction of nuclear Nfat1 (right) in HSCs lentivirally transduced with sPRrdm16 and flPRdm16. Plots, mean ± s.e.m.; n ≥ 14 fields of cells pooled from two biological replicates; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test. k, Nfat luciferase gene reporter activity in WT and in Prdm16−/− MEFs transfected with Mfn2, flPrdm16 or sPrdm16 constructs. Bars, mean ± s.d.; n = 3 experiments; *P < 0.05; one-way ANOVA with Dunnett’s post-hoc test.
