Journal: Science Advances

Article Title: Tumor gasdermin E acts as a rheostat to limit apoptotic cell survival and mutagenesis in anticancer immune environments

doi: 10.1126/sciadv.aeb1137

Figure Lengend Snippet: ( A ) Immunoblotting of SIINFEKL peptide–pulsed Neuro2a cell lines cocultured with activated OT-I T cells for 4 hours at ratios of T cells to target cells indicated. ( B ) Schematic representation of fluorescence-activated cell sorting (FACS) based on Caspase-3(DEVD)NucView and annexin V (AnnV) staining, applies to (C) to (E). Created in BioRender; B. Liu (2026), https://BioRender.com/y3nlv0g . ( C ) Rate of outgrowth from cells recovered from FACS sort, calculated on the basis of number of initially plated cells. Data were compiled from five independent experiments. Paired t test. ( D and E ) Quantification of nucleotide fragments with greater than three, four, five, or six single-nucleotide variants (SNVs) from various recovered populations as indicated. ( F ) The design of the ATM–fluorescence resonance energy transfer (FRET) reporter. Created in BioRender; B. Liu (2026), https://BioRender.com/jqdhdx7 . ( G ) Quantification of FRET loss signal from Neuro2a expressing the ATM-FRET reporter shown in (F). Neuro2a cells were SIINFEKL peptide pulsed and then coincubated with OT-I T cells for 6 hours at 10:1 T cell–to–target cell ratio. Data were pooled from three independent experiments. Unpaired t test. ( H ) In vivo growth of Neuro2a cells expressing the ATM-FRET sensor. ATM-FRET loss populations were sorted post BH3m treatment (1 μM ABT737 and 1 μM S63845 ). Cells were sorted for FRET-loss population at 2.5 hours, and 1 × 10 4 cells were injected into NSG mice ( n = 5 animals per group). Kaplan-Meyer survival curves. WT-base and GE KO–base refer to ATM-FRET–expressing cells that were not treated with BH3m. Log-rank (Mantel-Cox) test. * P < 0.05 and **** P < 0.0001. C3, active caspase-3.

Article Snippet: Cells were spun down and resuspended in DMEM supplemented with annexin binding buffer (99902, Biotium) and stained with annexin V-647 and/or Caspase-3(DEVD)NucView-488, as indicated in the text, for 10 min at 37°C.

Techniques: Western Blot, Fluorescence, FACS, Staining, Förster Resonance Energy Transfer, Expressing, In Vivo, Injection

Journal: Science Advances

Article Title: Tumor gasdermin E acts as a rheostat to limit apoptotic cell survival and mutagenesis in anticancer immune environments

doi: 10.1126/sciadv.aeb1137

Figure Lengend Snippet: ( A ) Coculture (1:1) of parental B16F10 cells expressing pBabe-Cerulean (blue) and B16F10 cells expressing GsdmD-C3 and mCherry (red) (see movie S1). ( B ) Times of individual cell shrinkage and cell membrane lysis, as in (A). Unpaired t test. ( C ) Apoptotic phase (shrinkage-lysis interval) from (B). One-way ANOVA. ( D ) Quantification of annexin V + SIINFEKL peptide–pulsed B16F10 [as in (B) and (C)] that remains fluorescent when coincubated with activated OT-I T cells at 4 hours. Two-way ANOVA. ( E ) Quantification of SIINFEKL peptide–pulsed B16F10 cell populations with ectopic caspase-3-activatable GSDMD (GD-C3) by fluorescence signal at 24 hours of coincubation with activated OT-I T cells. ( F ) As in (E) with Gsdme. [(E) and (F)] Paired t test. ( G ) Number of GCamp6s spikes observed from SIINFEKL peptide–pulsed Neuro2a cells, coincubated with activated OT-I T cells, 10:1 T cell–to–target cell ratio. Images were taken every 2 min for a duration of 3 hours immediately following the additional of OT-I T cells, and numbers of observable GCamp6s spikes were counted and plotted (see movies S2 to S4 and fig. S5C). Unpaired t test. ( H ) Design for (I) and (J). Created in BioRender; B. Liu (2026), https://BioRender.com/j67ipmw . ( I ) Bioluminescence signal intensity of Neuro2a in C57BL/6 animals ( n = 9 animals per group). Two-way ANOVA. ( J ) Kaplan-Meyer survival curves from C57BL/6 mice injected with Neuro2a cells ( n = 9 animals per group). ( K ) Design for (L). Created in BioRender; B. Liu (2026), https://BioRender.com/j67ipmw . ( L ) Bioluminescence signal intensity of Neuro2a in C57BL/6 animals (control, n = 9 animals; Gsdme −/− , n = 8 animals). ( M ) Kaplan-Meyer survival curves from C57BL/6 mice rechallenged with Neuro2a cells [ n = 5 animals per group; log-rank (Mantel-Cox) test]. ( N ) As in (M) with B16F10 [ n = 12 animals in naïve group, 14 animals in rechallenge group; log-rank (Mantel-Cox) test]. * P < 0.05, ** P < 0.01, and **** P < 0.0001. iv, intravenous; ip, intraperitoneal.

Article Snippet: Cells were spun down and resuspended in DMEM supplemented with annexin binding buffer (99902, Biotium) and stained with annexin V-647 and/or Caspase-3(DEVD)NucView-488, as indicated in the text, for 10 min at 37°C.

Techniques: Expressing, Membrane, Lysis, Fluorescence, Injection, Control