Fig 2: YTHDF1 is recruited by METTL3 to enhance SLC7A11 mRNA stability and translation. A and B The knockdown efficiency after 48 h transfection of YTHDF1 siRNA and negative control siRNA (siNC) in PC9 cells was confirmed by RT-qPCR and western blot. C and D The relative mRNA expression determined by RT-qPCR and the protein expression showed by western blot of SLC7A11 after YTHDF1 knockdown in PC9 cells. E SLC7A11 mRNA half-lives (t1/2) showed by RNA decay rates followed by RT-qPCR after YTHDF1 knockdown in PC9 cells. Data were collected at indicated timepoints (0 h, 3 h, and 6 h) with actinomycin D (Act D, 5 µg/mL) treatment. F RIP-qPCR revealed the binding enrichment of YTHDF1 to SLC7A11 in METTL3 stable knockdown and negative control PC9 cells. G The protein levels of SLC7A11 showed by western blot in YTHDF1 knockdown H322 cells with METTL3 overexpression, compared with control H322 cells. H Flow cytometry analysis of intracellular ROS levels in YTHDF1 knockdown H322 cells with METTL3 overexpression, compared with control H322 cells. I Flow cytometry analysis for apoptotic cell proportion (Q2 + Q3) by Annexin V-FITC/PI staining in H322 cells. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant

Fig 3: MRP1 inhibitor MK-571 shifts cellular thiol pools, further potentiating APR-246 efficacy Total intracellular glutathione (GSH + GSSG per 106 cells), as determined by GR re-cycling assay after 24-h MK-571 treatment in six different cell lines (n = 3, except HCT116 WT n = 2). *P = 0.014, Paired t-test. Each dot represents one cell line, see Fig EV6 for individual cell lines and n.Intracellular oxidized glutathione (GSSG per 106 cells) as shown by LC-MS in OVCAR-3 (R248Q TP53) cells at 24-h treatment with APR-246 +/- MK-571 (n = 3).Intracellular reduced glutathione (GSH per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24-h treatment with APR-246 +/- MK-571 (n = 3).Western blot analysis of MRP1 (Cell Signaling), xCT, and GAPDH of OVCAR-3 cells treated with APR-246 +/- MK-571 for 24 h.Western blot analysis of MRP1 (Cell Signaling), xCT, and GAPDH of HCT116 WT and R248W cells treated with APR-246 +/- MK-571 for 24 h.Intracellular cystine (CySS per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24 h treatment with APR-246 +/- MK-571 (n = 3).Intracellular cysteine (Cys per 106 cells) as shown by LC-MS in OVCAR-3 cells at 24 h treatment with APR-246 +/- MK-571 (n = 3). Data information: TP53 status is indicated. Data are represented as mean ± SEM. See also Fig EV6 and Appendix Fig S6. Source data are available online for this figure.

Fig 4: Inhibiting Xc- activity reduced intracellular GSH levels and potentiated bortezomib-induced cytotoxicity in myeloma cell lines and primary cells. (a) ANBL-6 cells were treated with 0.25 mm sulfasalazine (SASP) or 5 mm glutamate (Glu) for 16 h. Intracellular GSH was measured as described in Materials and methods. Data are mean and s.d. from triplicates of one representative of three independent experiments. Asterisk indicates statistically significant differences as compared with the control (two-way ANOVA, Turkey's multiple comparisons test, P<0.05). INA-6 (b) or ANBL-6 cells (c) were treated with the indicated doses of bortezomib (Bz) alone or in combination with 0.25 mm sulfasalazine for 24 h. Sulfasalazine treatment induced a significant decrease in the IC50 of bortezomib. INA-6 (d) or ANBL-6 cells (e) were treated with the indicated doses of bortezomib and 5 mm glutamate for 24 h. Glutamate treatment induced a significant decrease in IC50 of bortezomib. (f) Bortezomib-conditioned (Bz-cond.) and non-conditioned (Non-cond.) INA-6 cells were treated and analyzed as in (b). Sulfasalazine treatment induced a significant decrease in IC50 of bortezomib in both conditioned and non-conditioned cells. (g) SLC7A11 was targeted in INA-6 cells using CRISPR/Cas9. Clones were lysed and xCT levels were determined by immunoblotting. Bars give the quantified bortezomib-induced change of xCT levels. ERK1/2 was used as a loading control as GAPDH levels were reduced in xCT clones. Data are representative of at least three independent experiments. (h) Clones from (g) were treated with the indicated doses of bortezomib and 0.25 mm sulfasalazine for 24 h. Bortezomib displayed a significantly lower IC50 in xCT knockdown clones than in non-targeting control clones. (i) CD138+ plasma cells from a myeloma patient were treated for 3 days with bortezomib alone or in combination with 0.25 mm sulfasalazine. Sulfasalazine treatment induced a significant decrease in IC50 of bortezomib. The ScanR microscope was used to measure cell death. All IC50 values are in nm, and were calculated using non-linear regression. Extra sum-of-squares F-test was used to test for significant shifts in IC50 (P<0.05). (b–f) and (h) Mean and standard deviation from duplicates in one representative experiment of at least three independent experiments is shown. (i) Mean and standard deviation from duplicates in one experiment is shown.

Fig 5: YAP/TAZ transcriptionally upregulate SLC7A11 expression Combinatorial analysis of the genes upregulated in Sorafenib-resistant cells and the genes downregulated upon YAP/TAZ depletion uncovered 56 common genes, among which was SLC7A11.Quantitative RT-PCR analysis confirmed the dependency of SLC7A11 gene expression on YAP/TAZ. HLE cells were transfected with control siRNA (siCtrl) or siRNA against YAP/TAZ (siY/T) and cultured with DMSO or 6 µM Sorafenib for 18 h. RNA was extracted and analyzed by quantitative RT-PCR. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.SLC7A11 protein levels were upregulated by the exposure to Sorafenib, yet downregulated by siRNA-mediated depletion of YAP/TAZ. HLE cells were transfected with siCtrl or siY/T and cultured with DMSO or 6 µM Sorafenib for 18 h before harvest, followed by immunoblotting for YAP/TAZ and SLC7A1. GAPDH served as loading control. Results represent three independent experiments.siRNA-mediated ablation of YAP/TAZ significantly reduced SLC7A11 promoter activity, as determined by SLC7A11-promoter-luciferase reporter assay. HLE cells were transfected with SLC7A11-promoter firefly luciferase reporter construct and a constitutive-active Renilla luciferase reporter construct (pRL-CMV) and with siCtrl or siY/T. Relative luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega E1980). Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.A potential TEAD binding motif was predicted at – 400 bp within the promoter region of SLC7A11. PCR primers SLC7A11-CH1 (P1) and SLC7A11-CH2 (P2) were designed to examine the potential binding of transcription factors to the TEAD binding motif by chromatin immunoprecipitation (ChIP).Binding of YAP and TAZ to a DNA fragment containing the TEAD binding motif in the SLC7A11 promoter. ChIP was performed on HLE cell lysate with antibodies against YAP and TAZ and rabbit IgG as control. DNA fragments were amplified using the primers specific for TEAD binding motif in the SLC7A11 promoter region shown in (E). The non-coding region NC10 served as negative control, and the bona fide TEAD target gene CYR61 served as positive control. Data are shown as mean ± standard deviation (SD). Statistical significance was calculated using one-way ANOVA. Results represent three independent experiments.Knockdown of YAP/TAZ impairs cystine uptake either with or without Sorafenib treatment, and cystine uptake decreased with the exposure to Sorafenib. HLE-shLuc and HLE-shY/T cells were cultured with DMSO or 6 µM Sorafenib for 18 h, cystine-FITC was added to cells, and after incubation at 37°C for 30 min, intracellular Cystine-FITC levels were measured by flow cytometry using a 488 nm laser. Results represent three independent experiments.Colony formation assay showing that stable overexpression of SLC7A11 reversed Sorafenib-induced cell death in YAP/TAZ-deficient HCC cells. HLE-shLuc and HLE-shY/T were or were not transfected to overexpress SLC7A11 (SLC7A11 OE) and treated with DMSO or 6 µM Sorafenib for 2 weeks as indicated. Results represent three independent experiments.Representative images of immunohistochemical staining of SLC7A11 and YAP proteins in HCC samples from patients. Scale bars, 50 µm.Quantification of the immunohistochemical stainings shown in (I) revealed a positive correlation of YAP and SLC7A11 expression (N = 10). Statistical significance was calculated using Pearson correlation analysis. Source data are available online for this figure.