The effect of LOUP on PU.1 expression, myeloid differentiation, and cell growth. (A-C) qRT-PCR expression analysis for LOUP (left panels) and PU.1 (right panels). (A) Nontargeting (N) and LOUP-targeting (L) U937 CRISPR/Cas9 cell clones. Data are shown relative to N1 control. (B) Cord blood CD34⁺ HSPCs stably transfected with LOUP-targeting (shLOUP) or nontargeting (shControl) shRNAs by lentiviral transduction and grown in liquid culture with myeloid differentiation-promoting cytokines for 12 days. (C) K562 dCas9-VP64-stable cells infected with LOUP-targeting (#A1 and #A2) or nontargeting (Control) sgRNAs. (D) Trypan blue exclusion and manual cell counts for kinetics of cell growth (sgControl vs sgLOUP [#A1 and #A2]). (E) Representative flow cytometry results of CD11b myeloid marker expression. Cord blood CD34⁺ HSPCs stably transfected with either shLOUP shRNA or shControl shRNA by lentiviral transduction and grown in liquid culture with myeloid differentiation-promoting cytokines for 12 days. (F) Camco Stain Pak staining of shControl and shLOUP samples as described in panel E. Representative images were acquired with a Nikon Eclipse microscope (original magnification, ×60) and the SPOT Insight2 camera. Error bars indicate SD (n = 3). *P < .05; **P < .01; ***P < .001; ****P < .0001. See also supplemental Figure 4 and supplemental Table 3. n.s., not significant.