Enhanced IGFBP7 expression sensitizes APL cells to physiological concentrations of ATRA, and rhIGFBP7 potentiates ATRA-induced differentiation, inhibition of proliferation, and apoptosis in non-APL AML cells. For all experiments, AML cells were cultured in low serum condition and stimulated with PBS (Control/Ctrl), 300 µg/mL rhIGFBP7, 1.0 µM ATRA, or the combination (Combi) for 96 hours unless stated otherwise. Differentiation (membrane CD11b expression), proliferation, and apoptosis were measured using flow cytometry, and data are plotted as mean ± SEM. (A) Percentage of differentiation in NB4 cells, transduced with IGFBP7 shRNAs (#1 and #2) or scrambled (SCR) shRNA, after stimulation with 0.5 µM ATRA. P values were determined using 1-way ANOVA with post hoc Dunnett multiple-comparison test. (B-C) NB4 cells, transduced with control or IGFBP7-expressing (IGFBP7-OE) vectors, were stimulated with 0.01 µM ATRA. (B) Percentage increase in CD11b expression. (C) CFU assay (in duplicate), normalized against untreated control. (D) CD11b expression in HL60 cells, quantified relative to flow count beads. (E) Morphology of HL60 cells after stimulation for 72 hours, analyzed using a bright-field microscope (upper panels) and by May-Grünwald-Giemsa staining (lower panels). (F) Viability of AML cell lines after stimulation for 72 hours (HL60 and OCI-AML3 cells) or 120 hours (THP1 and KG1a cells) with rhIGFBP7, ATRA, or the combination, normalized against untreated control. (G) AnnexinV⁺ THP1 cells after stimulation for 120 hours. (H) Percentage of apoptotic (AnnexinV⁺ and 7AAD⁺) THP1 cells (left panel) or OCI-AML3 cells (right panel) after stimulation for 120 hours, quantified relative to flow count beads. Graphs are representative of ≥3 independent experiments. *P < .05, **P < .01, ***P < .001, ****P < .0001, 1- or 2-way ANOVA with post hoc Tukey’s multiple-comparison test, unless stated otherwise.