JAK/STAT5 pathway activation is required for IL-7-induced T-ALL cell viability and growth. (A) IL-7-starved TAIL7 cells were incubated with IL-7 for the indicated periods of time, and JAK-STAT5 pathway activation was analyzed by immunoblot. In the P-JAK1/3 panel, the upper bands correspond to P-JAK1 and the lower bands to P-JAK3. Results are representative of at least 2 independent experiments. (B) TAIL7 cells were nucleofected with pGL3-β-casein-firefly luciferase vector and pGL4-SV40-Renilla luciferase, followed by IL-7 stimulation for 24 hours. Luciferase activity from cell extracts was measured in a luminometer. STAT5 transcriptional activity was calculated as described in “Methods.” (C) Primary T-ALL or PDX cells were stimulated with IL-7 for 15 minutes or 2 hours, respectively, followed by immunoblot analysis of STAT5 activation. Data representative of 2 patients and 3 PDX analyzed. (D) HPB-ALL cells, stably transduced with STAT5A shRNA (shSTAT5) or scramble control (shSCR), were stimulated with IL-7 for 15 minutes and evaluated for STAT5 activation (P-STAT5) and total STAT5 expression. (E) Stably transduced HPB-ALL cells cultured for 72 hours in the indicated conditions were assessed for viability and cell growth. Results are representative of 3 independent experiments. Graphics represent average of triplicates ± standard error of the mean (SEM).