LNK depletion enhances colony forming potential and cytokine-induced STAT5 signaling in CD34⁺ HSPCs. CB-derived HSPCs were transduced with shRNAs to Luc or LNK and sorted for mCherry⁺ cells 48 hours posttransduction. (A-B) mCherry⁺ cells were either directly plated in methylcellulose (day 3) or cultured in cytokine-containing media for an additional 5 days (day 8) and subsequently plated in methylcellulose for CFC assay. CFU progenitor frequency was quantified after 12 to 14 days. (A) Representative CFU counts are shown per 500 CD34⁺ HSPCs. (B) Distribution of different types of colonies is shown. N = 3 CBs. Each symbol represents an individual plate; bars indicate mean values; error bars indicate plus or minus SD. *P < .05; **P < .01; ***P < .001 as determined by 2-way ANOVA followed by Šídák’s multiple comparisons. Representative colonies and full CFU plates are shown. (C) Purified mCherry⁺ CD34⁺ HSPCs were starved for 2 hours and stimulated with different concentrations of GM-CSF for 10 minutes. Cells were lysed and subjected to western blot analysis with indicated antibodies. Representative blots for cytokine-induced JAK/STAT activation are shown. Protein levels were quantified by the intensity of the bands then normalized to Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) controls, and the numbers are shown underneath the respective protein band. (D) Relative protein levels of LNK and phosphorylated proteins of individual CB samples after stimulation with 20 ng/mL GM-CSF is shown. N = 3 CBs. Each colored symbol and dashed line indicate an individual CB sample in each independent experiment. *P < .05; **P < .01; ***P < .001, calculated using paired 2-tailed Student t tests. GEMM, granulocyte, erythrocyte, monocyte, megakaryocyte CFUs.