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. 2020 Jul 29;8:703. doi: 10.3389/fcell.2020.00703

FIGURE 5.

FIGURE 5

Bergenin regulates the expression of ABCG2 and SLC2A9 in HK-2 cell lines. (A) HK-2 cell lines were treated with bergenin (10, 30, 50, or 100 μM) for 12 h, and cell viability was determined by the CCK8 assay. (B) HK-2 cell lines were pretreated with bergenin (0, 30, 50, or 100 μM) for 2 h, followed by treatment with 8 mg/dL uric acid (UA) for 10 h. Relative mRNA levels of ABCG2 and SLC2A9 were determined by RT-qPCR. (C) Representative western blot showing ABCG2, SLC2A9, and PPARγ protein levels. Protein levels were normalized to GAPDH. (D) Transcriptional activity of PPARγ, as determined by luciferase assay. (E) Cells were transfected with PPARγ siRNA or scrambled siRNA for 48 h. Cells were then pretreated with or without 50 μM bergenin for 2 h, followed by exposure to 8 mg/dL UA for another 10 h. Relative mRNA levels of ABCG2, SLC2A9, and PPARγ were determined by RT-qPCR. (F) Representative western blot showing ABCG2, SLC2A9, and PPARγ protein levels. Protein levels were normalized to GAPDH. (G) Cells were pretreated with bergenin (50 μM) or rosiglitazone (PPARγ agonist) for 2 h, followed treatment with 8 mg/dL UA for another 10 h, with or without GW9662 (PPARγ inhibitor). ABCG2, SLC2A9, and PPARγ protein levels were determined by western blotting. Protein levels were normalized to GAPDH. (H) Total p53 and acetylated p53 levels were measured by western blot analysis. Protein levels were normalized to GAPDH. Cytoplasmic and nuclear extracts were prepared for western blot analyses. Cytoplasmic protein levels were normalized to GAPDH, whereas nuclear protein levels were normalized to Lamin A/C. (I) Representative immunofluorescence images (magnification, ×400) showing p53 expression (green). Nuclei were stained with DAPI (blue). Scale bar = 50 μm. (J) Transcriptional activity of p53, as determined by luciferase assay. (K) Cells were pretreated with bergenin (50 μM) or the p53 inhibitor Pifithrin-β (PFT-β) for 2 h, followed by treatment with 8 mg/dL UA for another 10 h, with or without WR-1065 (p53 agonist). ABCG2, SLC2A9, and p53 protein levels were determined by western blot analysis. Protein levels were normalized to GAPDH. Data are presented as mean ± SEM. *P < 0.05 and **P < 0.01, n = 3.