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. 2023 May 8;13(9):2757–2773. doi: 10.7150/thno.81406

Figure 2.

Figure 2

HMGB1 is induced by RLDC treatment of cultured renal tubular cells. BUMPT cells were incubated with cisplatin for 7 h every day for four days or left untreated (Con or 0 μM), and collected at day 5. (A) Representative immunoblots of fibronectin (FN), HMGB1, and GAPDH (loading control). (B) Densitometric analysis of the immunoblots in A. (C) Immunofluorescence analysis of HMGB1 (green) translocation. The nuclei were stained with Hoechst (blue). The arrows indicate cytoplasmic HMGB1 in RLDC-treated cells. Scale bar = 20 μm; scale bar in magnified images = 10 μm. (D) Quantitative analysis of HMGB1 staining in BUMPT cells. (E) Immunoblots of HMGB1 in the nuclear and cytosolic fractions of untreated (Con) and RLDC-treated BUMPT cells. Histone 3 protein (H3) and GAPDH were used as internal controls for nuclear fractions and whole cell lysate, respectively. (F) Densitometric analysis of the immunoblots in E. (G) qRT-PCR analysis of Hmgb1, Myd88, Tlr2, and Tlr4 mRNAs. The expression of the target genes was normalized to GAPDH mRNA and expressed as fold change compared to control cells (Con). Quantitative data are expressed as mean ± SEM. N = 5. * P <0.05 vs. the control group (Con).