2C). STAT5 binding to the Socs2 gene promoter served as a positive control. Western blot Selleckchem ABT737 analyses confirmed the reduction of NOX4 in Stat5−/− MEFs (Supporting Fig. 2D). NOX4 and BIM levels were increased

in Stat5−/−/Stat5A MEFs compared with parental Stat5−/− MEFs, further supporting that STAT5 directly controls expression of these genes (Supporting Fig. 2E). Expression of Puma and Bim was STAT5-dependent and under GH control in MEFs (Supporting Fig. 3A). Western blot analyses confirmed the reduction of PUMA and BIM in Stat5−/− MEFs (Supporting Fig. 2D). Overexpression of STAT5A in Stat5−/− MEFs further increased Puma and Bim mRNA levels (Supporting Fig. 4A), and GH-dependent induction of Puma and Bim expression was observed in Stat5−/−/Stat5A MEFs but not in Stat5−/− MEFs carrying an empty control retrovirus (Supporting Fig. 4B). Tyrosine phospho-STAT5 was detected in GH-stimulated Stat5+/+ MEFs (Supporting Fig. 3C), and elevated levels were observed in Stat5−/−/Stat5A MEFs (Supporting Fig. 3D). Levels of phospho-p53 were also increased in Stat5−/−/Stat5A MEFs compared with

parental Stat5−/− MEFs (Supporting Fig. 2E). Puma as a p53 target gene might be regulated by STAT5/p53 signaling. One GAS motif was identified at position −605 in the Puma gene, and two conserved GAS motifs were identified at positions −3684 and −540 in the Bim gene (Supporting Fig. 4C). ChIP analyses in Stat5+/+ MEFs confirmed GH-induced STAT5 binding to these GAS motifs (Supporting Fig.

Carfilzomib mw 4C). Binding to the Socs2 gene promoter served as a positive control. To explore the mechanistic links between phospho-p53 and expression of a subset of p53 target genes, we analyzed Stat5−/− and Stat5−/−/Stat5A MEFs. Expression of Bax, Fas, Noxa, and Ataf was increased in Stat5−/−/Stat5A MEFs compared with Stat5−/− Phospholipase D1 MEFs carrying an empty control retrovirus (Supporting Fig. 5). Expression of the p53 gene was not changed in Stat5−/−/Stat5A MEFs compared with Stat5−/− MEFs. To determine whether ROS generation is under direct STAT5/NOX4 control, Stat5+/+ and Stat5−/− MEFs were cultured and assayed for ROS using DCF-DA and lucigenin. DCF fluorescence, an indicator of ROS, was stronger in Stat5+/+ MEFs than in Stat5−/− MEFs (Supporting Fig. 6A). Treatment with H2O2 further increased the production of ROS in Stat5+/+ MEFs compared with Stat5−/− MEFs (Supporting Figs. 6A and 7A). The lucigenin chemiluminescent assays established that STAT5 deficiency led to a reduced level of intracellular ROS in MEFs (Supporting Fig. 6B). Treatment of Stat5+/+ MEFs with diphenylene iodonium (DPI), a NOX inhibitor, reduced ROS levels (Supporting Figs. 6A and 7B). Although DPI inhibits several NOX members, NOX4 is the only one expressed at appreciable levels in liver tissue. This suggests that ROS in MEFs originates from NOX4.