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. 2021 Jul 19;12:720387. doi: 10.3389/fphar.2021.720387

FIGURE 3.

FIGURE 3

ICA alleviates fibrosis by inducing autophagy in HK-2 and NKR-49F cells (A) HK-2 cells were treated with HG and rapamycin for 48 h, then the expression of p62, Col I, and FN was determined by Western blot (B–F) The relative levels of BECN1, SQSTM1, COL1A1, ACTA2, and FN1 were detected by real-time PCR after HK-2 cells were stimulated with HG and various concentrations of ICA for 48 h ****p < 0.0001 vs NG; ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs HG (G–I) Protein expression of autophagy and fibrosis in HK-2 and NRK-49F cells after incubation with HG and ICA (2, 10, 50 μM) or TGF-β1 (5 ng/ml) for 48 h (J) The cell viability was analyzed in NRK-49F cells after incubation with HG and various concentrations of ICA for 48 h n = 6; ****p < 0.0001 vs NG, # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001 vs HG (K–L) The total apoptosis rates detection and analysis by flow cytometry after NRK-49F cells were treated with HG and ICA for 48 h (mean ± SD; n = 3; *p < 0.05, ****p < 0.0001) (M) HK-2 cells were infected with an LC3B-encoding adenovirus, treated with HG (30 mM), ICA (50 μM) or CQ (5 μM). Scale bar: 50 μM, original magnification: ×630. Representative immunofluorescence images are shown (N–R) HK-2 cells were transfected with siATG5 or NC, then treated with or without ICA (50 μM). The expression of ATG5, LC3, and Col I was evaluated and analyzed by the ChemiScope analysis software (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).