Extended Data Fig. 4: The role of SLC7A11 in p53^R175H-mediated inhibition of ferroptosis and promotion of tumor growth.

a, Identification of SLC7A11^-/- Cal-33 cells by Western blot analysis. b, Images of tumors isolated from nude mice implanted with Cal-33 Ctrl and SLC7A11^-/- cells. n = 8 tumors. c, Tumor weights for Cal-33 Ctrl and SLC7A11^-/- xenografts. n = 8 tumors. P value: <0.0001. d, Representative images of 4-HNE staining of Cal-33 Ctrl and SLC7A11^-/- xenograft tumors. Bar scale=20 μm. e, Quantification of 4-HNE staining using the immunoreactive score (IRS) system (n = 6 fields from 6 tumors). P value: 0.0004. f, Identification of p53^R175H-/- SLC7A11 overexpressing Cal-33 cells by Western blot analysis. p53^R175H-/- SLC7A11 overexpressing Cal-33 cells were made by transducing plenti6-SLC7A11/xCT-V5 lentivirus into p53^R175H-/- cells. g, Images of tumors isolated from nude mice implanted with Cal-33 p53^R175H-/- and p53^R175H-/- +SLC7A11 cells. n = 8 tumors. h, Tumor weights for Cal-33 p53^R175H-/- and p53^R175H-/- +SLC7A11 xenografts. n = 8 tumors. P value: 0.0008. i, Representative images of 4-HNE staining of Cal-33 p53^R175H-/- and p53^R175H-/- +SLC7A11 xenograft tumors. Bar scale=20 μm. j, Quantification of 4-HNE staining using the IRS system (n = 6 fields from 6 tumors). P value: <0.0001. k, Representative images of ferroptotic cell death in Cal-33 Ctrl, SLC7A11^-/-, p53^R175H-/- and p53^R175H-/-+ SLC7A11 cells treated with 10 μM erastin for 72 h. Red arrows indicate cells undergoing ferroptotic cell death. Bar scale=50 μm. The images shown are representative of 3 repeats with similar results. l, Cell death assay for Cal-33 Ctrl, SLC7A11^-/-, p53^R175H-/- and p53^R175H-/- + SLC7A11 cells treated with 10 μM erastin for 72 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. m, Cell death assay for Cal-33 Ctrl, SLC7A11^-/-, p53^R175H-/- and p53^R175H-/-+SLC7A11 cells treated with 0.85 mM TBH for 7 h in presence or absence of 5 μM ferr-1. n = 3 technical cell culture replicates. The experiment was repeated three times with similar results. Two-tailed Student’s t-test were used for statistical analysis. ***p < 0.001; Data (l, m) represent mean of three technical replicates. Data (c, e, h and j) represent mean + S.E.M.

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