Fig 2: ACSL4-induced ferroptosis promotes renal fibrosis in mice with CaOx kidney stones. (A) Overall docking plot of abemaciclib and ACSL4. (B) Body weight changes in mice (n = 5). (C–G) Measurement of the levels of BUN, Scr, GSH, CAT, and MDA in the serum of mice (n = 5). (H) HE, Von Kossa, Masson, and Perls staining to assess tubular damage, CaOx crystals, collagen fibrillation and iron deposition (n = 5). The scale bar represents 50 µm. (I) Western blot analysis showed the expressions of ACSL4, P53, SLC7A11, GPX4, fibronectin, and α-SMA in mouse kidney tissues and quantification by densitometry. (J) Immunofluorescence staining analysis of ACSL4, SLC7A11, GPX4, and α-SMA expressions in mouse kidney tissues and semi-quantitative analysis (n = 5). The scale bar represents 50 µm. (K) TEM revealed mitochondrial damage (indicated by red arrows) in mice with CaOx kidney stones. The scale bar represents 1 µm. Data were presented as mean ± SD; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; # p < 0.05, ## p < 0.01 vs. Gly group.

Fig 3: ATR phosphorylates USP20 and induces USP20‐dependent ferroptosis resistance. (A) Co‐IP assay reveals association between endogenous USP20 and ATR in Huh‐7 cells. Huh‐7 cells were harvested with RIPA lysis buffer. Co‐IP was performed using antibody as indicated. (B) The expression of ATR, P‐ATR, USP20, and SLC7A11 in Huh‐7/OXA and its parental cells. (C) Coimmunoprecipitation analysis of USP20 serine phosphorylation in Huh‐7 cells treated with ATR siRNA or siControl. (D) ATR phosphorylates S132 and S368 sites of USP20. (E) Inhibition of ATR by siRNA affected the turnover of USP20. Huh‐7 cells were treated siATR. Cells were then treated with CHX for the indicated time, and the expression of USP20 was analyzed by western blotting. (F) USP20 phosphorylation prolonged its half‐life. (G) HEK293T cells transfected with HA‐ubiquitin and Flag‐USP20 or indicated Flag‐USP20 mutant plasmids were treated ATR siRNA. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of USP20 was detected by HA antibody. (H) The relative cell viability of HCC primary cells treated with the indicated dosage of erastin. (I) Crystal violet staining of HCC primary cells treated with erastin. (Each group contained three replicates.) (J–L) Lipid ROS (J), GSH levels (K), and ferrous iron levels (L) were measured in HCC primary cells. (M) In vivo xenografts generated from primary HCC cells expressing an empty vector, USP20‐WT, USP20‐S132A/S368A, or USP20‐ S132D/S368D. 1 × 106 primary HCC cells were injected to the right dorsal flank of each mouse (n = 6). Tumor sizes were measured every 5 days until the end of the experiment. (N) Huh‐7 cells transfected with shUSP20 or shControl plasmids were treated with OXA for 12 h. Then Huh‐7 cells were treated with increasing concentration of erastin for 24 h. The relative cell viability of HCC cells was detected using CCK8 assay. (Each group contained three replicates.) (O) Huh‐7 cells transfected with shUSP20 or shControl plasmids. CCK8 assay showing the response of HCC cells were treated with erastin (20 μM) or RSL‐3 (5 μM) in the absence or presence of ferrostatin‐1 (1 μM), liproxstatin‐1 (1 μM), Z‐VAD‐FMK (10 μM), necrosulfonamide (0.5 μM), or 3‐methyladenine (250 μM) for 24 h. (P–R) Huh‐7 cells transfected with shUSP20 or shControl plasmids. Lipid ROS (P), GSH levels (Q) and ferrous iron levels (R) were measured in HCC cells. Results shown are representative of three independent experiments. Data are represented as mean ± SD of biological triplicates.*p value < 0.05, **p value < 0.01, ***p value < 0.001.

Fig 4: ACSL4-induced ferroptosis promotes COM-induced fibrosis in HK-2 cells. (A) Western blot analysis showed the expressions of ACSL4, GPX4, fibronectin and α-SMA after stimulation of HK-2 cells with different concentrations of COM and quantification by densitometry. (B) Cell viability of HK-2 under different concentrations of COM stimulation. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group. (C) Western blots validated ACSL4-siRNA knockdown efficiency. ** p < 0.01 vs. COM group. (D) Western blot analysis showed the expressions of P53, SLC7A11, GPX4, fibronectin and Collagen I after knockdown of ACSL4 and quantification by densitometry. (E) Immunofluorescence analysis of SLC7A11, GPX4 and α-SMA expressions in HK-2 cells after knockdown of ACSL4 and semi-quantitative analysis. The scale bar represents 50 µm. (F) Measurement of lipid peroxidation in HK-2 cells by the C11 BODIPY581/591 fluorescent probe. The scale bar represents 50 µm. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. Con-siRNA group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Con-siRNA + COM group. Data were presented as mean ± SD.

Fig 5: USP20 de‐polyubiquitylates SLC7A11. (A) Huh‐7 cells transfected with the indicated shRNA were treated with MG132 for 6 h before collection. SLC7A11 was immunoprecipitated with anti‐SLC7A11 and immunoblotted with anti‐HA. (B) Immunoblotting was used to detect the ubiquitination of SLC7A11 in HEK293T cells cotransfected with Myc‐SLC7A11, HA‐ubiquitin and Flag‐USP20 (wild type or C154A). (C) USP20 removed the ubiquitin chain of SLC7A11 in a time‐ and dose‐dependent manner. (D) HA‐WT, K6, K11, K27, K29, K33, K48, or K63 Ub were cotransfected with Myc‐SLC7A11 and Flag‐USP20 into HEK293T cells. After treatment with 10 μM MG132 for 6 h, cell lysates were subjected to ubiquitination assay and the ubiquitination level of SLC7A11 detected by HA antibody. (E). Immunoblotting to detect the ubiquitination of the SLC7A11 deletion mutants (FL, ΔN, and ΔN) in HEK293T cells cotransfected with Myc‐SLC7A11 (FL, ΔN, and ΔN), HA‐Ub and USP20. (F) A schematic diagram of SLC7A11 and its mutants. (G) Immunoblotting to detect the ubiquitination of SLC7A11 mutants (K4, K12, K30, K37, K41, and K43) in HEK293T cells cotransfected with Myc‐SLC7A11 mutants, USP20 and HA‐Ub.