Supplementary Figure 8: Mitochondria-associated mRNAs are selectively regulated by mTORC1 in erythroid cells.

(a) Representative cytospin preparations from CD34 + HSPCs, differentiated ProEs and granulocytes. Scale bars, 20 μm. The images represent one out of three independent experiments. (b) Granulocyte differentiation is characterized by progressively decreased expression of CD34 and increased expression of granulocyte-specific marker CD66b. Representative histograms for HSPCs, day 3 and 6 differentiated granulocytes are shown. (c) Granulocyte differentiation is characterized by loss of GATA2 expression and increased expression of granulocyte-specific genes DEFA1 and LTF, whereas erythroid differentiation is characterized by increased expression of the hemoglobin gene HBB. Results are mean ± s.e.m. of n = 3 independent experiments. (d) Polysome profiling of granulocytes treated with control (DMSO) or PP242 (2.5 μM) for 12 h. (e) qRT-PCR analysis of indicated transcripts in granulocytes treated with control (DMSO) or PP242. Results are mean ± s.d. of n = 3 independent measurements and shown as the percentage (%) of total mRNA of all fractions combined. (f,g) Inhibition of mTORC1 or depletion of TFAM had minimal effect on the expression of granulocyte-specific marker CD66b or signature genes. Results are shown as mean ± s.e.m. of n = 3 independent experiments and analyzed by a two-tailed t-test. (h) Analysis of Mac1 and Gr1 expression in BM of Mrp8-Cre⁻; Tfam^fl/fl (WT, n = 4) and Mrp8-Cre⁺; Tfam^fl/fl (KO, n = 4) mice (4 ∼ 5 weeks old). The representative FACS cytograms out of four independent experiments are shown. (i) Granulocyte-specific KO of Tfam had minimal effect on the frequency of BM Mac1⁺Gr1⁺ granulocytes. (j) Expression of Tfam was determined by qRT-PCR in Mac1⁺Gr1⁺ granulocytes from Mrp8-Cre⁻; Tfam^fl/fl (WT, n = 4) and Mrp8-Cre⁺; Tfam^fl/fl (KO, n = 4) mice. (k) Granulocyte-specific Tfam KO decreased mtDNA in Mac1⁺Gr1⁺ granulocytes. (l) Expression of Defa1 and Ltf mRNA remained unchanged in Mac1⁺Gr1⁺ granulocytes upon Tfam KO. Results are mean ± s.e.m. of n = 4 WT and n = 4 KO mice by a two-tailed t-test (i–l). ^∗∗P < 0.01, ^∗∗∗P < 0.001, n. s. not significant. See Statistics Source Data in Supplementary Table 8. (m) The wild-type (WT) and mutated (MUT) sequences of TOP-like motifs at the 5′UTRs of TFAM, PHB2 or ATP5D transcripts are shown.