Fig. 5

CTSB was identified as a crucial gene in FRA-induced HSFs ferroptosis. (A) KEGG pathway enrichment analysis of the DETs between the CTL and FRA (300 µM for 48 h)-treated groups. The red boxes indicate the signalling pathways of interest. (B) GO analysis of the DETs in different groups. The red boxes indicate the signalling pathways of interest. (C) Structural model of CTSB complexed with FRA. In the close-up view, the hydrogen bonds formed between the compound and the protein are depicted as dashed black lines, and the His111, His112, and His200 residues are involved in hydrogen bond interactions. (D) Immunofluorescence staining of CTSB and LAMP1 in HSFs incubated with or without FRA for 48 h (scale bar = 50 μm). (E) Quantification of the relative fluorescence intensity of CTSB. (F) The protein levels of ACSL4, SAT1, pro-CTSB and CTSB in HSFs treated with or without FRA for 48 h were analysed by WB. (G) Quantification of ACSL4, SAT1, pro-CTSB and CTSB protein levels based on WB analysis. (H) The mRNA expression of CTSB in HSFs treated with FRA (300 µM) for 24, 48 and 96 h was measured by qRT‒PCR. GAPDH was used as the internal reference gene. (I) WB analysis of the levels of MAPK signalling pathway proteins, including p-ERK/ERK, p‐JNK/JNK and p‐p38/p38, in HSFs treated with various concentrations of FRA (100 µM, 200 µM and 300 µM) for 48 h. (J) The p‐ERK/ERK, p‐JNK/JNK and p‐p38/p38 protein ratios were quantified based on WB analysis. The results are expressed as the means ± SDs (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ns = not significant