Fig. 5: PTENα restricts oxidative cell death.
From: PTENα functions as an immune suppressor and promotes immune resistance in PTEN-mutant cancer
a Microscopy analysis of H2O2 (500 μM) treated Mock or PTENα-expressing B16-Pten−/− cells. UT untreatment. Scale bar: 100 μm. b Mock or PTENα-expressing B16-Pten−/− cells were treated with 500 μM H2O2 and the percent cytotoxicity was assessed using LDH release assay (n = 4 cell cultures, mean ± SD, ****P < 0.0001). UT untreatment. c Mock or PTENα-expressing B16-Pten−/− cells were treated with 500 μM H2O2 in the absence or presence of 1 μM Fer1. Cells were stained with propidium iodide (PI), followed by flow cytometric analysis. All cells were gated. The death cell rates were used for statistical analysis (n = 3 cell cultures, mean ± SD, ****P < 0.0001). UT untreatment. d and e In vitro cytotoxicity assay for B16 cells. Mock or PTENα-expressing B16-Pten−/− cells pulsed with GP33–41 peptide were used as target cells. Effector cells and target cells were incubated at indicated ratios. Effector-uninfection refers to non-antigen-specific T cells and acts as a negative control. d Before incubation, CD8+ T cells were stained with CFSE, then the effector cells and target cells were incubated in the presence or absence of 20 mM NP-VE. 20 h post-incubation, the cells were harvested and subjected to PI staining. Death cells in the CFSE− cells were assessed by flow cytometric analysis (n = 3 cell cultures, mean ± SD, ns not significant, *P = 0.0188, ****P < 0.0001). Gating strategies were shown. e 12 h post-incubation, cells were harvested and stained with BODIPY-C11, followed by flow cytometric analysis. The gating strategy was identical to that in Fig. 4h. MFI of the target cells was used for statistical analysis (n = 3 cell cultures, mean ± SD, *P = 0.0207). f Mock or PTENα transfected Pten−/− B16 cells were subjected to the cancer vaccination model and subcutaneously injected in the presence or absence of 50 μl of 5 M NP-VE. Tumor volumes were monitored over time, and the statistical significances between the Mock groups were indicated (PTENα + NP-VE, n = 7 mice; other groups, n = 6 mice, mean ± s.e.m., *P = 0.0465, **P (d20) = 0.0015, **P (d20) = 0.0019). Statistical significance was assessed by two-tailed unpaired Student’s t test (b, e, f) or one (d) or two (c)-way ANOVA followed by Tukey’s multiple comparisons test. Data are representative of two (a–f) independent experiments. Source data are provided as a Source Data file. See also Supplementary Figs. 8 and 9.
