Fig. 2

a Top: Proliferation curve showing prolonged survival for BaF3 cells expressing either JAK2 p.G571S protein alone or both JAK2 p.G571S and STAT3 p.K370R proteins. Transfected BaF3 cells were cultured in media without IL-3 for 7 days and their proliferation measured every day using trypan blue. JAK2 p.V617F serves as positive control. Values represent the mean out of three replicates with essentially identical datasets (n = 3). Bottom: Immunoblot analysis of BaF3 cells expressing STAT3-WT, STAT3-p.K370R, JAK2-p.G571S, JAK2-p.V617F, or STAT3-p.K370R+JAK2-p.G571S. Depicted are p-STAT3 and p-STAT5 levels of the respective cells. Total STAT3, STAT5, as well as beta-ACTIN serve as loading controls (n = 2). b Top: Proliferation curve showing prolonged survival for BaF3/CRLF2-IL-7RWT cells expressing either JAK2 p.G571S alone or both JAK2 p.G571S and STAT3 p.K370R. Cells harboring both mutants show a significantly higher proliferation rate compared to cells expressing JAK2 p.G571S, starting at day 2 after IL-3 withdrawal (calculated by Student’s t test: p values are indicated). Cells were cultured as described in (a) (n = 3). Bottom: Immunoblot of BaF3/CRLF2-IL-7RWT cells harboring Empty Vector (EV), STAT3-WT, STAT3-p.K370R, JAK2-p.G571S, or STAT3-p.K370R+JAK2-p.G571S expression plasmids. Depicted are p-STAT3, p-STAT5, and p-AKT levels of the respective cells. Total STAT3, STAT5, AKT, as well as GAPDH serve as loading controls (n = 2). c Top: Cell cycle analysis showing an accumulation of >4n, as well as a reduction of cells in the G1-phase for STAT3 p.K370R expressing BaF3 cells, as compared to cells expressing STAT3-WT or STAT3 p.K370R+JAK2 p.G571S. p Values are indicated as calculated by Student’s t test (n = 3). Bottom: Immunoblot depicting the expression of the cell cycle related proteins CDC2 and Cyclin-B1/B2/A2 in BaF3 cells expressing either EV, STAT3-WT, STAT3-p.K370R, JAK2-p.G571S, or both mutant proteins. GAPDH serves as loading control