WWOX silencing results in increased expression of SMAD3 target genes. (A-C) Validation of increased gene expression of SMAD3-regulated genes in MCF10 (A) shWWOX subline (white bars) compared to Scr shRNA control (black bars) by Real Time qPCR. mRNA from three biological replicates of each stable cell line were used for quantitation, 18S rRNA was used as normalization control, (p < 0.01 for all genes). Further real-time PCR validation of SMAD3 target upregulation in WWOX-silenced (shWWOX-B, white bars) or Scr shRNA control (black bars) 184B5 normal breast cells (B) or MCF7 breast cancer cells (C). Significant upregulation was seen for all target genes in all WWOX-silenced samples with the exception of PTHLH expression in MCF7 cells. (D) ELISA assay for quantitation of ANGPTL4 protein concentration in culture media from each MCF10 subline after 72 hours in culture (p < 0.01). (E) ELISA assay for FST protein concentration in culture media from MCF10 sublines. (F) Reversion of SMAD3 target gene upregulation by inducible ectopic WWOX expression in MCF10 shWWOX-A cells. Real-Time PCR analysis of MCF10 shWWOX-A cells transiently transduced with a DOX-inducible WWOX expression system. With induction of ectopic WWOX expression (black bars) or without WWOX expression (white bars). 18S rRNA used as normalization control (p < 0.05 for all genes).