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. 2020 Sep 10;295(47):15870–15882. doi: 10.1074/jbc.RA120.015623

Figure 4.

Figure 4.

Ascorbic acid inhibits PCSK9 transcription in a Foxo3a-dependent manner and enhances LDLR transcription by activating SREBP2. HepG2 cells were transfected with control siRNA (si-NC, 25 nm) or siRNA against FoxO3a (si-FoxO3a, 25 nm) for 48 h, followed by treatment with AA (20 µm) or vehicle in fresh medium for 12 h. Expression of FoxO3a, PCSK9, and LDLR protein (A), and FoxO3a and PCSK9 mRNA (B) were determined by Western blotting and qRT-PCR, respectively. C and G, 293T cells were transfected with DNA for normal human PCSK9 promoter (phPCSK9), PCSK9 promoter with IRE mutation (phPCSK9-IREmut); LDLR promoter (phLDLR) or LDLR promoter with SRE mutation (phLDLR-SREmut) plus Renilla luciferase (for internal normalization), respectively. After transfection and indicated treatment overnight, activity of firefly and Renilla luciferases in cellular lysate were determined using the Dual-Luciferase Reporter Assay system. D and H, 293T cells were transfected with control siRNA (si-NC, 25 nm) or siRNA against FoxO3a (si-FoxO3a, 25 nm) or siRNA against SREBP2 (si-SREBP2, 25 nm) for 48 h, respectively. Cells were then transfected with DNA for normal human PCSK9 promoter (phPCSK9, D) or for normal human LDLR promoter (phLDLR, H). After transfection and indicated treatment overnight, activity of firefly and Renilla luciferases in cellular lysate were determined using the Dual-Luciferase Reporter Assay system. E and F, HepG2 cells were transfected with control siRNA (si-NC, 25 nm) or siRNA against SREBP2 (si-SREBP2, 25 nm) for 48 h, followed by treatment with AA (20 µm) or vehicle in fresh medium for 12 h. Protein levels of LDLR, SREBP2 precursor (p), and mature form (m) were determined by Western blotting (E). The normalized intensity of mature SREBP2 ((m) SREBP2)) versus β-actin was calculated. Expression of LDLR and SREBP2 mRNA was examined by qRT-PCR (F). I, HepG2 cells were pre-treated with actinomycin D (Act D, 10 nm) for 2 h and then treated with AA (20 μm) or vehicle for the indicated times. Expression of LDLR mRNA was determined by qRT-PCR. *, p <0.05; **, p < 0.01 versus corresponding controls or as indicated (n = 5); ##, p < 0.01 versus si-NC plus AA (n = 5).