Expression of autophagy-related genes in HSCs derived from P5 and 4-week-old mice. (A) Expression level of autophagy-related genes was compared between P5 neonatal and 4-week-old mice HSCs by RNA-seq. Graphs show expression value estimated based on RPKM (n = 4 in each condition; *P < .05 by Student t test). (B) Enrichment of gene sets KEGG_REGULATION_OF_AUTOPHAGY, POSITIVE RETULATION OF AUTOPHAGY, and NEGATIVE RETULATION OF AUTOPHAGY within differentially regulated genes in P5 neonatal and 4-week-old mice HSCs. (C) Autophagy activity in P5 neonatal and 8-week-old adult HSCs was assessed by CYTO-ID staining. Graph shows fluorescent intensity relative to the value of P5 HSCs pretreated without chloroquine. Data are presented as means ± SD (n = 3 in each condition, 2 independent experiment; *P < .05 by Student t test). (D) Atg7^fl/fl and Atg7^fl/fl; Vav-Cre HSCs were fixed and stained with LC3 antibody. Autophagy flux was calculated from the geometric fluorescence intensity (GeoMFI) of LC3 in the absence or presence of chloroquine²⁴  (The value of GeoMFI in the presence of chloroquine was divided by the value of vehicle). Data are presented as means ± SD (n = 3 in each condition, 2 independent experiments; *P < .05 by Student t test).