PGE₂ increases in vivo expression of Bcl-X, SOCS2, and SOCS3.

(A) AS-E2 cells transfected with Bcl-X-luciferase reporter were EPO deprived for 16 hours and were left unstimulated (control) or were stimulated with EPO (2 U/mL) or PGE₂ (1 μM) or both for 6 hours. The Bcl-X transactivation was measured as luciferase activity (see “Materials and methods”). Measured luciferase activities were corrected for β-galactosidase activities and expressed as percentage transactivation of unstimulated control cells. The mean values of 3 independent experiments are presented, and SEM values are indicated by bars. (B) EPO-deprived AS-E2 cells were cultured in 6-well plates and were left unstimulated (control) or were stimulated with EPO (2 U/mL) or PGE₂ (1 μM) or both for 1 hour. Total RNA was isolated and reverse transcribed with M-MLV reverse transcriptase and cDNA was used in a PCR reaction using specific primers for SOCS2, SOCS3, or β₂-microglobulin (β₂M) and HPRT as control. Shown is a representative experiment of 3 independent experiments. (C) SOCS2 and SOCS3 expression was quantified by real-time PCR analysis. Cycle threshold (CT) values of SOCS2 and SOCS3 were normalized against 18S expression. Shown are the relative SOCS expressions (mean ± SEM) of 3 independent experiments. The asterisk indicates significance versus unstimulated the group and the double asterisk indicates significance (P < .05) between the EPO and EPO plus PGE₂ group. (D) Western blot analysis of cytosolic cell extracts of AS-E2 cells that were pretreated with or without PGE₂ (1 μM) and stimulated with or without EPO (2 U/mL) for the indicated periods, using antibody against SOCS3. This blot was reprobed using an antibody against actin to confirm equal loading. Shown is a representative blot of 3 independent experiments.