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. 2021 Jan 4;283(6):3272–3280. doi: 10.1074/jbc.M708033200

FIGURE 1.

FIGURE 1

SARS-CoV N protein specifically up-regulates Smad3-mediated transcriptional response of TGF-β.A, N protein enhances TGF-β-induced CAGA-luciferase expression in a dose-dependent manner. HPL1 cells were co-transfected with CAGA-luciferase reporter (0.5 μg) and pcDNA3.1-N (0.1, 0.3, or 0.5 μg). At 24 h post-transfection, the cells were treated with 50 pm TGF-β. After 20 h, the cells were harvested for determination of luciferase activity. B, N protein enhances TGF-β-induced 3TP-luciferase expression in a dose-dependent manner. HPL1 cells were co-transfected with 3TP-luciferase reporter (0.5 μg) and pcDNA3.1-N (0.1, 0.3, or 0.5 μg). C, N protein synergizes with Smad3 to induce CAGA-luciferase expression. HPL1 cells were co-transfected with CAGA-luciferase (0.5 μg), pCS2-Myc-Smad3 (20 ng), and pcDNA3.1-N (0.5 μg). D, N protein has no effect on the ARE-luciferase expression. HPL1 cells were co-transfected with ARE-luciferase reporter (0.5 μg) plus FoxH1 (0.25 μg) and pcDNA3.1-N (0.1, 0.3, or 0.5 μg). E, N protein has no effect on the BRE-luciferase expression. HPL1 cells were co-transfected with BRE-luciferase reporter (0.5 μg), pCMV5-FLAG-OAZ (0.25 μg), pCMV5-BMPRIB(QD)-HA (0.1 μg), and pcDNA3.1-N (0.1, 0.3, or 0.5 μg). F, N protein enhances the TGF-β-induced expression of CAGA-luciferase in wild-type (WT) MEFs, but has no effect on CAGA-luciferase expression in Smad3-/- MEFs. The endogenous expression of Smad2 (S2) and Smad3 (S3) in WT MEFs and Smad3-/- MEFs were detected by anti-Smad2/3 immunoblotting. The asterisks indicate a statistically significant difference (**, p < 0.01). RLU: relative luciferase units.