Fig 2: TalaA treatment-induced transcriptome alteration.The SW480 cells were treated with two concentrations of TalaA for 12 h. A Volcano plot showing the differences in RNA expression after 5.0 µM TalaA treatment. VPL means low concentration (5.0 µM) TalaA treatment; VPC means no chemical treatment (DMSO Control). B The volcano plot showing the differences in RNA expression after 10.0 µM TalaA treatment. VPH means high concentration (10.0 µM) TalaA treatment; VPC means no chemical treatment (DMSO control). C Through KEGG-enrichment analysis, it was found that the ferroptosis pathway molecules were up-regulated by 5.0 µM TalaA. D KEGG-enrichment analysis showed that the ferroptosis pathway molecules were up-regulated by 10.0 µM TalaA. E The heatmap data showed 5.0 µM TalaA resulted in gene expression alteration of ferroptosis-correlated molecules including FTL, SAT2, ALOXE3, GSS, ALOX12, HMOX1, ACSL5, and so on. F The heatmap data showed 10.0 µM TalaA resulted in gene expression alteration of ferroptosis-correlated molecules including FTL, SLC7A11, HMOX1, ALOXE3, SAT1, SAT2, GSS, ACSL5, ALXO12, PCBP1, MAP1LC3P, and so on.

Fig 3: xCT-dependent ROS clearance mediates starvation resistance. Both mRNA (a) and protein (b) expression of the glutamate/cystine antiporter xCT were elevated in GSCs with high Gln/Glu ratios (JHH520 and 407) (loading control = ß actin) (n = 3, significance was calculated with a one-way ANOVA, * significantly decreased (p < 0.05) compared to JHH520, # significantly decreased (p < 0.05) compared to 407). (c) Relative GSH concentrations of L-Gln-starved cells (compared to controls in full medium) assessed with 1H-NMR spectroscopy of metabolic extracts (n = 3, significance calculated with one-way ANOVA). (d) ROS accumulation in L-Gln-starved and control cells measured by means of DCF fluorescence intensity (n = 3). (e) Immunoblotting verification of shRNA-based xCT knockdown and ZEB1 expression in shxCT and pLKO.1 (control) cells (loading control = ß actin). (f) Soft agar colony formation in shxCT and pLKO.1 (control) cells (n = 3). (g) Soft agar colony formation in JHH shxCT cells cultured in the presence or absence of L-Gln (n = 3). (h) Soft agar colony formation in GBM1 cells starved for L-Gln in either the presence or absence of 3 mM NAC. For all soft agar assays, representative pictures of NBT-stained colonies and quantifications of three assays are shown (n = 3, scale bar equals 1 cm). For all assays, the data are presented as mean ± SD. Significance was calculated with a one-sided t-test if not specified elsewhere, * p < 0.05. Abbreviations: ctrl, control; CFU, colony-forming units; Gln, glutamine; GSH, glutathione; NAC, N-acetylcysteine; n.s., not significant; ROS, reactive oxygen species. Supplementary Figure 2 (S2) corresponds to Figure 3b, Supplementary Figure 3 (S3) corresponds to Figure 3e.

Fig 4: The pro-proliferative effect of xCT in human melanomas.a Western immunoblots on protein lysates from several stable clones that have been transfected with either empty vector or exogenous xCT cDNA in two melanoma cell lines (SK-mel2 and SK-mel187) with the lowest endogenous xCT expression (Fig. 1a). b Cellular growth was measured on stable xCT or empty vector clones derived from SK-mel2 and SK-mel187 cells. Viable cells were counted using trypan blue exclusion method

Fig 5: Expression of xCT in human melanoma cell lines and tumors.a Western immunoblots were conducted to assess xCT protein expression from one normal human melanocytes and a panel of human melanoma cell lines. Two out of nine melanoma cell lines demonstrated relatively low levels of endogenous xCT protein (SK-mel2 and SK-mel187). Relative expression of xCT was presented as a fold change compared to normal melanocytes. b Transient transfection of empty vector and exogenous xCT cDNA in HEK293T cells was performed, followed by western analysis at 48 h post-transfection. GAPDH was used as loading control. c An example of xCT expression in human melanoma tumor specimens at various stages. Quantitative RT-PCR was performed to evaluate mRNA expression of xCT in tumor samples. In the bar graph (top panel), the values were presented as the mean ± SEM of three independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001. Normalization of xCT mRNA to GAPDH levels showed an increase of xCT as the disease progressed. N.S., normal skin, C.N., congenital nevi, P.M., primary melanoma, IT.M., in-transit melanoma, N.M., nodal metastatic melanoma, L.M., lymph node metastatic melanoma. Bottom panel: western immunoblots were conducted to assess levels of xCT protein expression. Relative expression of xCT was presented as a fold change compared to normal skin. N.S., normal skin, C.N., congenital nevi, P.M., primary melanoma, IT.M., in-transit melanoma, N.M., nodal metastatic melanoma, L.M., lymph node metastatic melanoma. d Immunohistostaining of xCT on normal skin and tumor sections from stages II and III melanoma patients