p53-MnSOD axis is critical for ex vivo expansion of HSCs by VPA. (A) Effect of VPA and p53 siRNA treatment on p53 and MnSOD protein levels in the TNCs cultured as indicated for 4 days. (B) Effect of p53 inhibition on MnSOD transcript levels assessed by quantitative reverse transcription polymerase chain reaction on the TNCs cultured for 4 days. mRNA levels were represented as fold change relative to mRNA levels of cells cultured with cytokines alone within each experiment that was performed in triplicate (n = 6). (C-D) Effect of MnSOD siRNA on the percentage and absolute number of CD34⁺CD90⁺ cells generated after 4 days of incubation (n = 5). (E-F) Effect of MnSOD siRNA on the mitochondrial mass (E) and ROS levels (F) in CD34⁺CD90⁺ cells cultured for 4 days. Graphs represent fold change of median fluorescence intensity of MitoTracker Green and MitoSOX, relative to the corresponding values observed in CD34⁺CD90⁺ cells cultured with VPA within each experiment (n = 7). (G) Model of the mechanisms underlying the ex vivo expansion of HSCs. The dotted arrows indicate possible mechanisms activated by VPA resulting in mitochondrial network remodeling. “n” is the number of biological replicates. Error bars with standard error of the mean. ****P ≤ .0001; ***P ≤ .001; *P ≤ .05 were determined by 2-way ANOVA for panels B-C, negative-binomial models for panels D and I, and unpaired Student t test for E-F.