Figure 6.

 NONO is required for PCDH19 effect on ERα-mediated gene expression. (A) MCF-7 cells were transfected with NONO or control siRNA for 24 h and subsequently co-transfected with expression plasmids (control, wild-type or mutant Myc-PCDH19) in conjunction with 3× ERE TATA luc reporter plasmid. Cells were cultured in charcoal-stripped medium for 16 h and then for 6 h in the presence or absence of 10 nM E2. Data are expressed as relative luciferase activity ± SD from three or more independent experiments. *P < 0.05 comparing +E2 versus –E2 SCR vector control, *¹P < 0.05 comparing +E2 SCR vector control versus  +E2 NONO siRNA vector control, *²P < 0.01 comparing -E2 SCR vector control versus -E2 SCR WT, **³P < 0.01 comparing –E2 SCR WT versus –E2 NONO siRNA WT, **⁴P < 0.01 comparing +E2 SCR WT versus  +E2 NONO siRNA WT, **⁵P < 0.01 comparing –E2 SCR MT versus –E2 SCR WT, *⁶P < 0.05 comparing +E2 SCR MT versus  +E2 SCR WT, *⁷P < 0.05 comparing +E2 NONO siRNA MT versus  +E2 SCR siRNA MT. (B) Western blot of MCF-7 cell extracts expressing wild-type or mutant Myc-PCDH19 were probed with anti-Myc antibody. Endogenous NONO was detected using anti-NONO antibody and β-Actin was used as a loading control. The level of NONO was measured relative to β-actin signal by the ImageJ program and the level of NONO knockdown for each treatment relative to SCR control (fixed at 1) is shown.