Supplementary Figure 7: Effects of mTORC1 hyperactivation or TFAM overexpression on mitochondria and erythropoiesis.

(a,b) Quantification of Western blot shown in Fig. 7d, e. Results are mean ± s.e.m. of n = 3 experiments. (c) Quantification of Western blot shown in Fig. 7l. Results are mean ± s.e.m. of control (Veh, n = 4), PP242 (n = 4) and Torin1 (n = 4) treated mice. Differences relative to day0 (a), DMSO (b), or Veh (c) were assessed using a one-way ANOVA. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. (d) Western blot of TFAM and PHB2 in human primary erythroid cells treated with rapamycin (10 nM, 100 nM and 1 μM), PP242 (10 μM) or Torin1 (1 μM) for 5d. (e) Quantification of Western blot in d. (f) Rapamycin treatment resulted in dose-dependent decreases in mtDNA. Results are mean ± s.e.m. of n = 3 experiments. Differences relative to DMSO were assessed using a one-way ANOVA; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 (e,f). (g) Validation of shRNA-mediated depletion of TSC1 and TSC2 in human primary erythroid cells. (h) Quantification of Western blot in g. Depletion of TSC1 or TSC2 increased MMP (i), impaired erythroid maturation (j), and induced apoptosis (k). Results are mean ± s.e.m. of n = 3 experiments. Differences relative to shNT were assessed using a one-way ANOVA; ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 (i–k). (l,m) Hematopoietic-selective Pten KO led to a significant increase in mitochondrial mass (l) and decrease in BM CD71⁺Ter119⁺ erythroid cells (m). Results are mean ± s.e.m. of control (Mx1-cre⁺, Pten^fl/+; n = 3) and Pten KO (Mx1-cre⁻, Pten^fl/fl; n = 3). (n) Validation of TFAM overexpression by Western blot. The quantified TFAM protein expression is shown on the bottom as mean ± s.e.m. of three experiments. (o) TFAM overexpression led to increased mtDNA. (p–t) TFAM overexpression in human primary erythroid cells increased mitochondria mass (p), MMP (q), partially rescued the differentiation defects (r), apoptosis (s) and expression of erythroid genes (t) upon PP242 (2.5 μM) or Torin1 (0.25 μM) treatment. Results are mean ± s.e.m. of n = 3 experiments and analyzed by a two-tailed t-test (l,m,o–t). ^∗P < 0.05, ^∗∗P < 0.01, n. s. not significant. See Statistics Source Data in Supplementary Table 8. Unprocessed original scans of blots are shown in Supplementary Fig. 9.