Oncogenic potential of IL7R-RRI is IL-7– and γc-dependent. (A) TM and EJM cysteine–lacking mutations found in the literature. *TM regions predicted by TMPred (http://www.ch.embnet.org/software/TMPRED_form.html) are shown inside the boxes. (B) Ba/F3 cells stably expressing IL7R-WT, IL7R-RRI, IL7R-P2, or the empty vector (pMIG) cultured with or without mouse IL-7 (mIL-7; 5 ng/mL). The number of viable GFP⁺ cells per 1000 PercP-labeled beads (BD, Franklin Lakes, NJ) was determined by flow cytometry. (C) Ba/F3 cells stably expressing IL7R-WT or IL7R-RRI were starved for 4 hours (without fetal bovine serum, IL-3, or IL-7), stimulated with different concentrations of mIL-7 over 15 minutes, and used to detect phosphorylated STAT5 (pSTAT5) by intracellular flow cytometry. (D) Ba/F3 cells (blue bars) or γc-knockout Ba/F3 cells (red bars) were transduced with pMIG empty vector, IL7R-WT, IL7R-RRI, or IL7R-P2 and cultured with or without mIL-7 (5 ng/mL). The number of viable GFP⁺ cells per 1000 beads was determined by flow cytometry after 48 hours. (E) HEK293T cells were transiently transfected with different combinations of IL7RA and γc constructs in conjunction with JAK3 and Renilla luciferase expression vectors plus the STAT3/5-responsive luciferase reporter construct. Medium was replaced 16 hours after transfection. Eight hours later, IL-7 or vehicle was added, and the cells were cultured for 12 hours before luciferase was measured in a plate reader. Firefly luciferase was normalized by Renilla levels. Data are representative of at least 2 independent experiments. Results are the mean ± standard deviation (SD) of triplicate samples. Data were analyzed by using analysis of variance (ANOVA) and Tukey post hoc test. ***P < .001.