Long-term HSC alterations in a hyperglycemic environment. HSC-SLAMs isolated from 4-month-old control and db mice, assayed after 48 hours of in vitro culture in the presence or absence of BSO. (A) Experimental scheme. (B) Real-time polymerase chain reaction analysis after culture showing the relative expression of the oxidative response gene Nqo1, Hmox1, and Gadd45. Graph indicates the relative percentage expression ± standard deviation (SD) (n = 2 performed in sextuplets). (C) Mean percentage ± SD of cells presenting FOXO nuclear localization (n = 5, with 50 cells measured per condition per experiment). (D) Relative percentage ± SD of colony number obtained in methylcellulose assay for each condition (n = 2 performed in triplicate). (E-G) RNA sequencing on control and db HSC-SLAMs cultured with and without BSO (n = 3 for each condition). (E) Principal component analysis (PCA) visualization of the first 2 principal components of all expressed genes. (F) Hierarchically clustered heatmap of differentially expressed genes (n = 2105; fold >1.5; empirical Bayes t test; P < .05) with statistically enriched GO biological processes (left) and example genes associated with prototypical oxidative response (right). (G) GO-Elite heatmap of paired relative enrichment for GO biological processes among upregulated genes. (H) Quantification of γH2AX foci (n = 3, with 50 cells measured per condition per experiment). Two-way ANOVA with Sidak’s post hoc test; *P < .05; **P < .01; ****P ≤ .0001; # P < .05; ### P < .001.