Fig. 7

CTSB inhibition attenuated the effect of FRA on HSFs ferroptosis in vitro. (A) Immunofluorescence staining of CTSB and LAMP1 in HSFs pretreated with or without CA-074-me (20 µM) for 2 h and then incubated with FRA (300 µM) for 48 h (scale bar = 50 μm). (B) Quantification of the relative fluorescence intensity of CTSB. (C) The protein levels of Pro-CTSB, CTSB, ACSL4, SAT1 and GPX4 under the indicated treatments were determined by WB. (D) The mRNA levels of SAT1 and CTSB in HSFs subjected to the indicated treatments were determined by qRT‒PCR. GAPDH was used as the internal reference gene. (E) MDA concentration in HSFs under the indicated treatments was assessed using an MDA assay kit. (F) Representative images of FerroOrange staining (scale bar = 50 μm) and C11-BODIPY staining (scale bar = 50 μm) in different treatment groups. (G) Quantitative analysis of the relative fluorescence intensity of ferrous iron and lipid peroxide. (H) Representative images of MitoTracker staining (scale bar = 50 μm) and JC-1 staining (scale bar = 50 μm). (I) Quantitative analysis of the relative fluorescence intensity of the MitoTracker Red area and the aggregate/monomer ratio. (J) Representative images of the collagen gel contraction assay of HSFs under the indicated treatments on day 3. The dashed white lines indicate the areas of collagen gel. (K) Quantitative results of the collagen gel contraction assay. (L) WB results of the expression of α-SMA, COL1 and COL3 in HSFs after the indicated treatments. The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001