Figure 1.
PAR rapidly accumulates following progestin stimulation. (A) PAR levels (green) increase in T47DM cells treated with R5020 for 15 min. Levels of PARP-1 (left panels) and phosphorylated PR Ser400 (right panels), as a measure of cells responding to R5020, are shown in red. Bar, 10 μm. (B) Elevation in PAR levels (solid line), as measured by PAR-capture ELISA following R5020, is inhibited by 3AB (dashed line); hormone causes a concomitant decrease in cellular NAD levels (solid line, bottom panel), which is also 3AB-dependent (dashed line, bottom panel). (C) PARP-1 knockdown by specific shRNA was confirmed by mRNA expression (histogram) and Western blotting (inset). R5020 induced higher mRNA levels from the MMTV-luc transgene (D) as well as from c-fos, DUSP1, and EGFR (E), all of which are abrogated in PARP-1 knockdown cell lines. (**) P < 0.01. Error bars represent the SEM. (F) The percentage of the cell population residing in S phase 24 h following R5020 was determined in PARP-1 knockdown and control cell lines.