CD9 blocking affects STAT5 activation and impedes self-renewal. (A) pYSTAT5 levels determined by intracellular flow cytometry staining of CD9-low (CD9 MFI < median) and CD9-high (CD9 MFI > median) expressing FLT3-ITD⁺ NPM1⁺ patient BM cells (n = 5). (B) Flow cytometric analyses of pYSTAT5 levels in IgG- or aCD9-treated JAK2^V617F BM cells after respective stimulations for 20 minutes. (i) Differences of pYSTAT5 levels (aCD9 minus IgG) (n ≥ 3/genotype, mean ± SEM). (ii) Representative histograms of pYSTAT5 signal of HSC/MPP1 cells unstimulated (us) or after TPO stimulation; antibody isotype-stained (Ctrl) cells are shown as control. (C-E) Colony formation assays of wt and JAK2^V617F mouse BM cells either treated with IgG or aCD9 (5 µg/mL). (C) (i) Representative pictures of colony formation and (ii) fold change in colony numbers (n ≥ 5/genotype and condition, mean ± SEM). (D-E) Quantification of LSK cells (n ≥ 5/genotype and condition, mean ± SEM) after aCD9 or IgG treatment (D) and representative FACS plots of c-kit/Sca-1 (i) and CD11b expression in JAK2^V617F cells (ii) after aCD9 or IgG treatment (E). Levels of significance were calculated using a paired Student t test (A-B), and an unpaired Student t test (C-D). *P < .05; **P < .01; ***P < .001.