Figure 2. FIEL1 promotes TGFβ signaling.... | Rockefeller University Press

Figure 2.

$Figure 2. FIEL1 promotes TGFβ signaling. (A and E) SMAD reporter assays. 293T cells were cotransfected with Cignal SMAD dual luciferase reporter plasmids along with empty, FIEL1, Con shRNA, or FIEL1 shRNA. 24 h later, cells were treated with TGFβ for 2–16 h. Cells were collected and assayed for luciferase activity to evaluate SMAD promoter activity. Data represent mean values ± SEM (n = 3; *, P < 0.05 compared with empty, Student’s t test). (B and F) 293T cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ treatment (0–2 ng/ml) for 1 h. Cells were then collected and immunoblotted. Cell lysates were immunoprecipitated using SUMO antibody before SMAD2, 3, and 4 immunoblotting (n = 2). (C and G) 293T cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ treatment (2 ng/ml) for up to 1 h. Cells were then collected and nuclear/cytosol fractions were isolated prior to immunoblotting. (D and H) MRC5 cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ dose course treatment for an additional 18 h. Cells were then collected and immunoblotted (n = 2). (I) MRC5 cells were seeded in 35-mm glass bottom dishes before being transfected with CON shRNA or FIEL1 shRNA for 48 h before TGFβ treatment for an additional 30 min. Cells were then fixed and immunostained with α-SMAD3.The nucleus was counterstained with DAPI and F-actin was counterstained with phalloidin (n = 3). Bar, 10 µm.$

FIEL1 promotes TGFβ signaling. (A and E) SMAD reporter assays. 293T cells were cotransfected with Cignal SMAD dual luciferase reporter plasmids along with empty, FIEL1, Con shRNA, or FIEL1 shRNA. 24 h later, cells were treated with TGFβ for 2–16 h. Cells were collected and assayed for luciferase activity to evaluate SMAD promoter activity. Data represent mean values ± SEM (n = 3; *, P < 0.05 compared with empty, Student’s t test). (B and F) 293T cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ treatment (0–2 ng/ml) for 1 h. Cells were then collected and immunoblotted. Cell lysates were immunoprecipitated using SUMO antibody before SMAD2, 3, and 4 immunoblotting (n = 2). (C and G) 293T cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ treatment (2 ng/ml) for up to 1 h. Cells were then collected and nuclear/cytosol fractions were isolated prior to immunoblotting. (D and H) MRC5 cells were transfected with empty, FIEL1, CON shRNA, or FIEL1 shRNA for 48 h before TGFβ dose course treatment for an additional 18 h. Cells were then collected and immunoblotted (n = 2). (I) MRC5 cells were seeded in 35-mm glass bottom dishes before being transfected with CON shRNA or FIEL1 shRNA for 48 h before TGFβ treatment for an additional 30 min. Cells were then fixed and immunostained with α-SMAD3.The nucleus was counterstained with DAPI and F-actin was counterstained with phalloidin (n = 3). Bar, 10 µm.

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