Fig 2: BAP1 regulates disulfidptosis through the inhibition of SLC7A11-mediated cystine uptake.A Validation of BAP1 and SLC7A11 dual-overexpressed UMRC6 cell line. B, C SLC7A11 overexpression counteracts the protective role of BAP1 on UMRC6 cells upon glucose deprivation. D SLC7A11 overexpression enhances the actin cytoskeleton proteins tailing under non-reducing conditions in UMRC6-BAP1 cells upon glucose starvation. E Validation of SLC7A11 knockout in UMRC6-EV, -BAP1, and -C91A cells. F Knockout of SLC7A11 saves glucose starvation-induced cell death in UMRC6-EV and -C91A cells. G, H Erastin (10 μM) treatment saves disulfidptosis in UMRC6-EV and C91A cells upon glucose deprivation. I Cystine deprivation saves glucose starvation-induced cell death in UMRC6-EV and -C91A cells, while 2 mM cystine treatment enhances cell death in UMRC6-EV, -BAP1, and -C91A cells. J Cystine at 2 mM aggravated disulfide stress in UMRC6-BAP1 cells cultured in glucose-free and glucose-containing medium. The above treatments were terminated when glucose starvation caused significant death of UMRC6-EV cells.

Fig 3: SLC7A11 plays an important role in the growth and invasion of osteosarcoma cells and is the key downstream gene of MLX.A WB analysis of MLX and SLC7A11 in 143B (left) and SJSA1 (right) upon MLX knockdown. B–E The measurement of Cysteine, GSH, ROS and lipid peroxidation level upon overexpressing SLC7A11 in MLX-silenced 143B cells. Data are represented as mean ± SD, n = 3. Unpaired t-test was used. F, G Relative growth curves of 143B (F) and SJSA1 (G) cells stably transduced with nontargeting scrambled control shRNA (shCont) or two SLC7A11 shRNAs (shSLC7A11#1 and shSLC7A11#2). Data are represented as mean ± SD, n = 3. Unpaired one-way ANOVA test followed by Dunnett’s test was used. Cell invasion upon knockdown of shMLX and/or overexpression SLC7A11 in 143B (H) and SJSA1 (I) cells. Data are represented as mean ± SD, n = 3. Unpaired t-test was used. ***: P ≤ 0.001, **: P ≤ 0.01, *: P ≤ 0.05, n.s.: not significant.

Fig 4: SNF2L maintains GSH homeostasis by regulating SLC7A11 expression.A Flowchart illustrating the TMT-based quantitative proteomics approach by mass spectrometry used in this study. B Volcano plot displaying differentially expressed proteins between SNF2L-deficient and parental MDA-MB-231 cells identified by mass spectrometry (n = 3). C List of the top 10 most strongly differentially expressed proteins between SNF2L-deficient and parental MDA-MB-231 cells identified by mass spectrometry. D–G Relative expression of SNF2L and SLC7A11 proteins was analyzed by Western blotting in the MDA-MB-231 (D), HT-1080 (E), Huh-7 (F) and H1299 (G) cell lines, with β-actin used as the loading control (n = 3). H Relative expression of SNF2L and SLC7A11 was analyzed by Western blotting in HCCLM3 cells, and β-actin was used as the loading control (n = 3). I Heatmap of RNA-seq expression data showing differentially regulated SLC7A11 genes in SNF2L-deficient and parental HT-1080 cells (n = 3). J The relative mRNA levels of SLC7A11 were measured by RT-qPCR in the indicated cells (n = 3). K The relative mRNA levels of SLC7A11 were measured by RT-qPCR in parental HCCLM3 cells and HCCLM3 cells stably expressing SNF2L (n = 3). L Diagram showing the regulatory network of GSH metabolism by the SLC7A11 gene. M Relative cystine uptake capacity was measured in SNF2L-deficient and parental MDA-MB-231 cells. N Relative cystine uptake capacity was measured in SNF2L knockdown and parental H1299 cells. Error bars are the mean±SD, all data are representative of at least three biologically independent experiments. p values were calculated using two-tailed unpaired Student’s t tests (J, K, M) or one-way ANOVA with Tukey’s multiple comparisons test (N). *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: xCT suppression by shRNA inhibits proliferation and tumorigenicity of H520 in vitro and in vivo. a Cell proliferation assays reveal significant growth inhibition induced by xCT knockdown in H520 (n = 4). b Evidence for xCT knockdown in H520 cells to affect the amino acids consumption and excretion in media (n = 4). c The knockdown of xCT significantly decreases the glucose consumption in H520 cells. d The knockdown of xCT significantly decreases the lactate production in H520 cells (n = 4). e The knockdown of xCT promotes more oxidative intracellular condition in the H520 cells (n = 6). f The knockdown of xCT significantly decreases the glutamine dependency in H520 cells (n = 4). g Knockdown of xCT significantly reduces the H520 cell invasion compared with the H520_Ctrl by matrigel cell invasion assay (n = 3). h The effect of xCT knockdown on tumorigenicity in nude mice. Both H520_Ctrl (left flank) and H520_xCT_KD (right flank) cells were injected subcutaneously into the nude mice (n = 10). Tumor burden was monitored twice a week by caliper. Tumor size was calculated as 3.14 × (Min)2 × (Max)/6, where Min and Max were from (length, width, and depth of tumor measurements). All the tumors (mm3) were measured in both flanks of 10 mice