MC5R deletion significantly alters cell cycle-associated transcriptional profile in HSPCs following irradiation exposure. (A-C) LSKs sorted from the BM of WT and MC5R^–/– mice 15 days after 5.0 Gy TBI were subjected to RNA-seq analysis (n = 3). Those with a fold change >1.5 and P-value < .05 were defined as differentially expressed genes (DEGs). (A) Experimental scheme. (B) Scatter plot showing DEGs in the LSKs from the BM of irradiated WT and MC5R^–/– mice. Representative DEGs are indicated. (C) Heatmap of significantly changed genes in LSKs from the BM of irradiated WT and MC5R^–/– mice. (D) GSEA of the RNA-seq data showing proliferation- and quiescence-associated enrichment, cell metabolism-associated enrichment, and protein synthesis-associated enrichment in LSKs from irradiated WT and MC5R^–/– mice. (E) qRT-PCR analysis of the relative expression of cell cycle-associated genes in LT-HSCs sorted from the BM of WT and MC5R^–/– mice 15 days after 5.0 Gy TBI (n = 3). (F) Flow cytometric analysis of mitochondrial mass in LSKs and LT-HSCs from the BM of WT and MC5R^–/– mice 15 days after 5.0 Gy TBI using MitoTracker Green staining (n = 6). Representative flow cytometric plots (left). (G) Flow cytometric analysis of mitochondrial membrane potential in LSKs and LT-HSCs from the BM of WT and MC5R^–/– mice 15 days after 5.0 Gy TBI by tetramethylrhodamine methyl ester (TMRM) staining (n = 6). (H) Flow cytometric analysis of the protein synthesis rate in LSKs and LT-HSCs from the BM of WT and MC5R^–/– mice 15 days after 5.0 Gy TBI by O-propargyl-puromycin (OP-Puro) incorporation analysis (n = 6). Representative flow cytometric plots are shown in the left. (E-H) Unpaired t test (two-tailed). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. MFI, mean fluorescence intensity.