Figure 6.

A, Pyruvate kinase (PK) activity was determined in lysates from 293T cells pretreated with ethanol (Vehicle), JAK inhibitor AG490 (AG490, Calbiochem, 50 μm), or Src inhibitor PP1 (PP1, Calbiochem, 10 μm). Cells were then were left untreated (gray bars) or treated with PRL (100 ng/ml for 20 min, white bars). Absolute values of PKM activity in the absence of PRL in vehicle, AG490, and PPI-treated cells were comparable (data not shown). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). B, PK activity was determined in lysates from 293T cells transfected with shCON, shRNA targeting JAK2 (shJAK2), or shRNA targeting Tyk2 (shTyk2) and either left untreated (gray bars) or treated with PRL (100 ng/ml for 20 min; white bars). Efficacy of these shRNAs (80–90% knockdown) have been previously reported (34). Absolute values of PKM activity in untreated cells transfected with different shRNA constructs were comparable (data not shown). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test). C, Lactate levels in the lysates from cells used in panel B were determined. D, PK activity was determined in lysates from 293T cells transfected with vector control (pcDNA3) or vectors for expression of JAK2 (wild type or TEL-JAK2 fusion or V617F mutant). Cells were then were left untreated (gray bars) or treated with PRL (100 ng/ml for 20 min, white bars). Asterisks denote statistical significance of obtained differences (P < 0.05 by Student’s t test).