PUM1 and PUM2 regulate FOXP1 expression in CD34⁺HSPCs and leukemic cells. (A) Immunoblot analysis of the indicated proteins in CD34⁺ HSPCs transduced or not (nt) with the indicated shRNA-encoding vectors at day 4 post-transduction. Actin was used as loading control. (B) RT-qPCR analysis of hFOXP1 mRNA in shRNA-transduced CD34⁺ cells at day 4 post-transduction. Results are normalized to GAPDH mRNA expression, and expressed relative to shCtrl-expressing cells (n = 3). (C) RT-qPCR analysis of hPUM1, hPUM2 and hFOXP1 transcripts in the indicated human stem/progenitor subpopulations (see Figure 1A). Results are normalized to GAPDH expression, and expressed relative to expression in HSC (n = 3). (D) Immunoblot analysis of the indicated proteins in shRNA-transduced MOLM-14 cells at day 3 post-transduction. Actin was used as loading control. (E) RT-qPCR analysis of hFOXP1 mRNA in shRNA-transduced MOLM-14 cells at day 3 post-transduction. Results are normalized to GAPDH expression and expressed relative to shCtrl-expressing cells (n = 3). (F) RT-qPCR analysis of FOXP1 expression in AML patient samples grouped according to FAB classification, and in CD34⁺ cells from healthy donors. Each symbol represents data from a single patient. CD34⁺: 11 samples; AML₀: 4 samples; AML₁: 9 samples; AML₂: 17 samples; AML₃: 6 samples; AML₄: 6 samples; AML₅: 4 samples. Results are normalized to GAPDH, HPRT and Cyclophilin A mRNA levels. (G) Correlation between PUM1 or PUM2 and FOXP1 mRNA expressions in AML samples. r = Pearson correlation’s coefficient. Data are expressed as mean ± SEM. ns: not significant, * P < .05; **P < .01; ***P < .001 [Mann-Whitney test in (F); Student t test in (B, C, E)].