Fig. 6

TGF-β interference with BMP signaling is enhanced in cells lacking Wdr68 expression.

A) Isolation of Wdr68/Dcaf7 knock-out C2C12 cell sublines and expression levels in growth medium (GM) versus differentiation medium (Diff). Panel A1) Lanes 1 and 4, Wdr68 protein was detected in the control NT1 cells. Lanes 2 and 5, Δwdr68-5 lacks wildtype Wdr68 protein expression. Lanes 3 and 6, Δwdr68-9 lacks wildtype Wdr68 protein expression. Panel A2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel A3) pYap1 levels did not differ substantially between lanes. Panel A4) Total Yap1 levels did not differ substantially between lanes. B) pSmad1/5 induction was not substantially altered in Δwdr68-5 or Δwdr68-9 sublines. Panel B1) pSmad1/5 levels in control (NT1) or Δwdr68-5 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B2) β-tubulin expression was used as a loading control and did not differ substantially between lanes. Panel B3) pSmad1/5 levels in control (NT1) or Δwdr68-9 (5) cells after 1 hour of exposure to 0, 1, 10, or 100ng/mL BMP4 in DM. Panel B4) β-tubulin expression was used as a loading control and did not differ substantially between lanes. C) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids and induced with 0, 1, 10, or 100ng/mL BMP4 in GM. No significant differences were found between control and deletion sublines. Representative experiment shown from at least 3 independent trials. D) Transient transfection of NT1, Δwdr68-5, and Δwdr68-9 sublines with BRE-Luc and SV40-Renilla plasmids, induced with 10ng/mL BMP4, and then challenged with 0, 0.1, 1.0, or 10ng/mL TGF-®1. At 10ng/mL TGF-®1 interference with BRE-Luc activity was significantly greater in the Δwdr68-5 and Δwdr68-9 sublines relative to NT1 controls (* = p < 0.002). Representative experiment shown from at least 3 independent trials. E-H) Immunofluorescence detection of pSmad1/5 in prim-12 stage zebrafish embryos raised at 32°C. E) wildtype sibling embryo. F) wdr68^{hi3812/hi3812} mutant embryo. G) DMSO-treated wildtype sibling. H) ISL-treated wildtype sibling.