JAK2 activity is required for CDC25A up-regulation. (A) JAK2^V617F-expressing FDC-P1 EPOR cells were treated with AG490 (50μM), an inhibitor of JAK2, harvested at indicated time, and subjected to Western blot analysis for CDC25A. (B) Left, JAK2^WT and JAK2^V617F-expressing FDC-P1 EPOR, HEL, and SET-2 cell lines were treated with AG490 (50μM, 1 hour) and subjected to Western blot analysis for the cell cycle regulators CDC25A, p27^Kip1, cyclin A, and CDC25C. Right, Western blot analysis of P-STAT5, STAT5, P-JAK2, and JAK2 was performed in JAK2^V617F-expressing FDC-P1 EPOR cell lines in response to AG490 treatment. (C) Left: JAK2^V617F-expressing FDC-P1–EPOR and HEL cell lines were treated with JAK2 inhibitor II (50μM, 1 hour), an inhibitor of JAK2, and subjected to Western blot analysis for CDC25A. Right: Western blot analysis of P-STAT5, STAT5, P-JAK2, and JAK2 was performed in JAK2^V617F-expressing FDC-P1 EPOR cell lines in response to JAK2 inhibitor II treatment. β-actin levels were used as control. Western blots are representative of at least 3 independent experiments.