Loss of SRC-3 significantly increases mitochondrial biogenesis and metabolism in HSCs. (A) Workflow of the microarray analysis. (B) Transcriptomic profiling of LSKs from WT and SRC-3−/− BM. Red-colored box (left) shows the number of upregulated genes and blue-colored box (right) shows the number of downregulated genes. (C) GO enrichment analysis of upregulated genes in LSKs after SRC-3 knockout. Shown are the top 10 enriched (P < .05) GO terms. (D) KEGG pathway enrichment analysis of upregulated genes in LSKs after SRC-3 knockout. Shown are the top 10 enriched (P < .05) pathways. (A-D) Microarray data are obtained from 1 experiment with 2 biological replicates (pooled from 4 mice per group). (E) Forward-scatter (FSC-A)/side-scatter (SSC-A) analysis of LT-HSCs, ST-HSCs, and MPPs from WT or SRC-3−/− BM (n = 10 mice per group). (F) Representative TEM images of mitochondria in LSKs from WT and SRC-3−/− BM. (G) Mitochondrial mass in MPs, LSKs, and LT-HSCs from WT and SRC-3−/− BM detected by flow cytometry with MTG staining (n = 5 mice per group). MFI, mean fluorescence intensity. (H) Representative TOMM20 immunofluorescence staining in LSKs from WT and SRC-3−/− mice. Scale bar, 5 μm. (I) Western blot analysis of mtDNA-coded proteins (MT-ND3, COX1, and MT-ATP6) in WT and SRC-3−/− LSKs (n = 5 mice per group were pooled). (J) Mitochondrial membrane potential of MPs, LSKs, and LT-HSCs from WT and SRC-3−/− BM determined by flow cytometry with DilC1(5) staining (n = 5 mice per group). (K) Oxygen consumption rate (OCR) of LSKs obtained from WT and SRC-3−/− mice (n = 3 mice per group). *P < .05, **P < .01.
