Figure 3∣. Ectopic expression of STAT3 or STAT5B mutants promotes growth in KAI3 cells under limiting IL2 concentrations.

(a) Quantification of the percentage of GFP⁺ cells post transduction of KAI3 cells with each STAT3 mutant observed in tumour samples. Transduced cells were cultured in regular IL2 concentrations for 4 days, then switched to limiting (25 IU ml⁻¹) IL2 concentrations. Day 0 = 4 days post transduction. Each data point shows the means ± s.d. of two biological replicates. The average values for the % of GFP⁺ cells at day 0 were 6.9, 3.2, 6.0, 2.8, 4.1, 4.3 and 3.2% for EV, STAT3-WT, S614R, G618R, Y640F, D661Y or A702T transduced cells, respectively. (b) Quantification of the percentage of GFP⁺ cells post transduction of KAI3 cells with each STAT5B mutant observed in tumour samples as in a. Means ± s.d. of two independent experiments with replicates are shown (n = 4). The average values for the % of GFP⁺ cells at day 0 were 4.8, 6.8, 5.9, 5.7, 8.2 and 6.7% for EV, STAT5B-WT, E579K, N642H, Y665F or I704L-mutant transduced cells, respectively. The percentage of GFP⁺ cells for each sample is normalized to day 0 in both a,b. (c) P-STAT5(Y699) and STAT5 protein expression levels in KAI3 cells transduced with STAT5B mutants are shown by western blot. Six days post transduction, the GFP⁺ population of STAT5B-mutant transduced cells was isolated by FACS, and then placed into culture medium with limiting (25 IU ml⁻¹) concentrations. Whole-cell lysates were collected 11 days post transduction. One experiment representative of three experiments is shown.