Figure 2

Loss of MBNL1 leads to elevated endocardial-produced TGFβ3 levels. Stage 14 AVC explants were transfected with or without MBNL1 siRNA. At 18 hrs medium was added to intact explants (A) or explants where the myocardium has been removed (B). Supernatants were collected after 20 hrs of conditioning and assayed by ELISA. The mean levels of secreted TGFβ proteins as percentages of the mock level + the standard errors of the means are shown. (A) TGFβ3, but not TGFβ2, levels were elevated in the supernatants from MBNL1 siRNA-treated explants. (B) TGFβ3 levels were elevated in supernatants from MBNL1 siRNA-treated explants in which the myocardium has been removed prior to conditioning. Immunofluorescence with an anti- TGFβ3 antibody was also performed on mock- (C, C’) or MBNL1 siRNA-transfected (D, D’) stage 14 AVC explants fixed after 38 hrs in culture. Bright field images of the endocardial monolayer in mock-transfected (C) and MBNL1 siRNA-transfected (D) explants correspond to immunofluorescence images for the same fields (C’, D’). (E) Stage 14 AVC explants were transfected with or without MBNL1 siRNA. After 38 hrs in culture, the myocardium was removed, RNA was harvested, and TGFβ3 levels were evaluated by real-time RT-PCR. TGFβ3 transcript levels did not change in response to MBNL1 knockdown in AVC endocardial cells. Error bars denote 95% confidence intervals. An asterisk indicates a significant difference from mock-transfected explants (P ≤ 0.05).