Fig. 7: PTENα maintains protein synthesis of peroxidases.

a Mock or PTENα expressing Pten^−/− B16 cells were treated with 100 μM H₂O₂ for indicated hours, followed by immunoblot analysis with anti-eIF2α and anti-phospho-eIF2α antibodies. Gray values of total eIF2α and phosphorylated eIF2α were determined, and the relative ratio of phosphorylated eIF2α to total eIF2α was indicated. b In vitro phosphatase assay of FLAG-tagged PTENα on phosphorylated eIF2α. Phosphorylation level of eIF2α was assessed by immunoblot analysis with anti-phospho-eIF2α antibodies. c ³⁵S-metabolic labeling analysis of Ptenα^+/+ and Ptenα^−/− MEFs treated with 100 μM H₂O₂ or 500 nM TG for 3 h. Gray values of autoradiographs were determined and used for statistical analysis (pooled samples, n = 3 independent experiments, mean ± SD, **P = 0.0020, ***P = 0.0007). d and e Puromycin incorporation assay of Mock or PTENα expressing Pten^−/− B16 cells treated with 100 μM H₂O₂ for indicated hours. Puromycin-labeled proteins were identified with confocal fluorescence microscopy (d) and immunoblot analysis (e). Scale bars, 100 μm. UT untreatment. f Ptenα^+/+ and Ptenα^−/− MEFs were treated with 100 μM H₂O₂, followed by mass spectrometry analysis. Proteins that were significantly decreased in Ptenα^−/− MEFs were analyzed using DAVID with Gene Ontology (GO) terms. g Mock or PTENα expressing Pten^−/− B16 cells were treated with 100 μM H₂O₂ for indicated hours, and expression level of GPX4 was assessed by immunoblot analysis with anti-GPX4 antibody. Gray values of GPX4 and α-tubulin were determined, and the relative ratio of GPX4 to α-tubulin was indicated. h Flow cytometry analysis of Mock or PTENα expressing Pten^−/− B16 cells treated with H₂O₂ (100 μM) for indicated hours and stained with DCFDA. Mean fluorescence intensities of DCFDA were used for statistical analysis (n = 3 cell cultures, mean ± SD, ****P < 0.0001). All live cells were gated. UT, untreatment. i Endogenous Gpx4 expression in B16-Pten^−/− cells was knockdown using shRNA targeting Gpx4 or scramble shRNA. Then the cells were transfected to express Mock or PTENα and subjected to cancer vaccination model. The tumor volumes were monitored over time (Scramble, n = 5 mice; shRNA, n = 6 mice, mean ± s.e.m., *P(d24) = 0.0238, *P(d26) = 0.0158). Statistical significances between scramble groups were shown. Statistical significance was assessed by a two-tailed unpaired Student’s t test (c, h, i). Data are representative of two (a, b, d, e, g–i) independent experiments or pooled from three (c) independent experiments. Source data are provided as a Source Data file. See also Supplementary Fig. 10.