p53 inhibition impairs expansion of HSCs with VPA by increasing ROS levels. (A) Transcript levels of MnSOD and Sestrin 2 in TNCs cultured for 4 days (n = 6). (B) Total and acetylated levels of p53, p21, and p-p38 were determined by western blot analysis. GAPDH (representative blot) was used as an internal control (n = 3). Relative fold change for p53, p21, and p38 in VPA-treated cells compare with those treated with cytokines alone was 2.58, 24.9, and 0.12, respectively. ImageJ was used to quantify WB images and values were normalized to GAPDH. (C-D) Effect of pifithrin α (Pα) on the percentage (C) and absolute number of CD34⁺CD90⁺ cells (D) cultured for 4 days (n = 8). Graphs represent fold change of the percentage of CD34⁺CD90⁺ cells treated as indicated relative to those treated with VPA within each experiment. (E) Effect of p53 inhibition on mitochondrial mass of CD34⁺CD90⁺ cells expanded for 4 days. Graphs represent fold change of MFI of MitoTracker Green in CD34⁺CD90⁺ within each experiment (n = 6). (F) Relative mitochondrial to nuclear DNA ratio of purified CD34⁺ cells from cultures treated for 4 days (n = 6). Graphs represent fold change of the mtDNA/nDNA ratio of CD34⁺ cells treated as indicated relative to those treated with cytokines alone within each experiment. (G) Effect of p53 activity inhibition on ROS levels in CD34⁺CD90⁺ cells expanded for 4 days. Graphs represent fold change of MitoSOX (MFI) in CD34⁺CD90⁺ cells within each experiment (n = 6). (H) Oxygen consumption rates measured by seahorse analyzer in CD34⁺ cells expanded for 4 days (n = 3). Experiments in H were performed in triplicates and values were normalized within each experiment. (I) Percentage of human CD45⁺ cells in the bone marrow of NSG mice 16 weeks posttransplantation with grafts derived from 4 different donors (n = 6). (J-K) Effect of nutlin 3a and NAC on the percentage of CD34⁺CD90⁺ cells expanded for 4 days (n = 5). (L-M) Effect of nutlin 3a or NAC on the mitochondrial mass (L) and mitochondrial ROS (M) of CD34⁺CD90⁺ cells (n = 6). Graphs in panels J-L represent fold change of the percentage of CD34⁺CD90⁺ cells treated as indicated relative to those expanded with VPA within each experiment. “n” is the number of biological replicates. Error bars with SEM, ****P ≤ .0001; ***P ≤ .001; **P ≤ .01; *P ≤ .05 were determined by negative-binomial models for panels E and I, unpaired Student t test for panels A, E, and G, 2-way ANOVA for panels C, F, J-M.