Knockdown of HSPA9 in human hematopoietic progenitor cells alters their erythroid differentiation. (A) Representative immunoblot of HSPA9 protein in CD34⁺ cells transduced with shRNAs targeting control genes (empty or luciferase) or HSPA9 after 7 days in erythroid culture conditions. (B) Quantification of HSPA9 protein levels in cells normalized to the luciferase shRNA (n = 2-5). (C) Representative flow cytometric plots for CD71 (transferrin receptor) and glycophorin A (GpA) expression in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each quadrant. CD71⁻/GpA⁻ cells are the most immature, CD71⁺/GpA⁻ are intermediate, and CD71⁺/GpA⁺ are the most mature cells.⁸  (D) Quantification of CD71⁺ cells (n = 4; *P < .0001). (E) Representative flow cytometric plots for CD15 and CD66 expression in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each quadrant. CD15⁻/CD66⁻ cells are the most immature, CD15⁺/CD66⁻ are intermediate, and CD15⁺/CD66⁺ are the most mature cells. (F) Quantification of CD15⁺ cells (n = 6). (G) Representative flow cytometric plots for CD41a and CD42b expression in control and HSPA9-knockdown cells. Numbers represent the percentage of cells in each quadrant. (H) Quantification of CD41a⁺ cells (n = 2). (I) Transduced cells were plated in methylcellulose medium containing puromycin, and the number of CFU-G plus CFU-GM colonies and BFU-E colonies were counted on day 12 (n = 2; *P = .04; **P = .003; ***P < .001). Pictures of representative CFU and BFU colonies are shown below the appropriate shRNA construct. Hemoglobin appears red in the representative BFU-E colonies; empty mean no shRNA sequence was inserted into the pLKO.1 vector. LUC indicates luciferase; and NS, not significant. All data represent the means ± SD.