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

Journal: Journal of Virology

Article Title: Foot-and-Mouth Disease Virus Exhibits an Altered Tropism in the Presence of Specific Immunoglobulins, Enabling Productive Infection and Killing of Dendritic Cells ▿

doi: 10.1128/JVI.02180-10

Figure Lengend Snippet: FMDV infection of moDC is closely associated with cell death and reduced ability to stimulate FMDV-specific T cell proliferation. (A) MoDC were exposed to medium, O1K-Cad2, or O1K-Cad2 IC at an MOI of 10 for 6 h. Phase-contrast microscopy captured the high levels of dying cells, including some exhibiting classical apoptotic morphology (high power; right). (B) MoDC were exposed to medium, O1K-Cad2, or O1K-Cad2 IC at an MOI of 10 for 1 to 8 h. Samples of each culture were labeled with antibodies against FMDV NS protein 3A or with annexin V conjugated to APC, and PI. Death was defined as staining with annexin V, PI, or both. The data presented are relative to medium-only values; R = 0.929 (Pearson's correlation coefficient) for the linear relationship between the proportion of cells positive for FMDV NS proteins and the proportion dying. (C) Purified CD4+ T cells from an FMDV-vaccinated animal were added to autologous moDC at a ratio of 1 DC to 100 T cells, with either medium, a sample of the antigen used for vaccination, purified O1K-Cad2, or purified O1K-Cad2 in IC at an MOI of 5. After 5 days, [3H]thymidine was added to each well and incubated overnight before harvesting and liquid scintillation counting of the cells for incorporated radioactivity. Pokeweed mitogen (PWM) was used as a positive control, and in all cases, counts exceeded 350,000 cpm (not shown). A representative data set is presented as cpm ± SD. *, P = 0.02; Student's t test. All 3 replicates using cells from 2 different animals were significant at this level or above.

Article Snippet: To detect dying cells in moDC cultures, 5 × 10 5 cells were pelleted by centrifugation and resuspended in annexin V binding buffer (MBL, Nagoya, Japan).

Techniques: Infection, Microscopy, Labeling, Staining, Purification, Incubation, Radioactivity, Positive Control

Journal: Scientific Reports

Article Title: Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains

doi: 10.1038/s41598-017-06024-2

Figure Lengend Snippet: S. aureus -induced platelet cell death. ( a ) MFI of platelet-bound DIOC6(3) (b) Annexin V binding (c) PMP concentration or (d) fold increase in active caspase-3 expression were analyzed by flow cytometry after 30 min of exposure to Tyrode’s buffer (NS), TRAP, staphylococcal strains [clinical strains (SaB), S. condimenti (Sc) or S. aureus ATCC 43866 (ATCC43866)] or the A23187 apoptosis inducer as a positive control. The data (mean ± SEM; n = 8 experiments) are expressed as the AU ( a ), % expression ( b ) or fold increase compared with NS ( d ). The PMP concentration (mean ± SEM; n = 6–27 experiments) ( c) is expressed as microparticles/µl. *p < 0.05, **p < 0.01, ****p < 0.0001 (one-way ANOVA with repeated measures and the Bonferroni post hoc test; stimulated vs . unstimulated, NS).

Article Snippet: In brief, after stimulation, platelets were resuspended in Annexin Binding Buffer (MabTag, Oldenburg, Germany) for immunostaining.

Techniques: Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Positive Control

Journal: Scientific Reports

Article Title: Acetylsalicylic acid differentially limits the activation and expression of cell death markers in human platelets exposed to Staphylococcus aureus strains

doi: 10.1038/s41598-017-06024-2

Figure Lengend Snippet: Effect of ASA and fluvastatin on S. aureus -induced platelet cell death. ( a ) The MFI of platelet-bound DIOC6(3) ( b ) Annexin V binding ( c ) PMP concentration or ( d ) fold increase in active caspase-3 expression were analyzed by flow cytometry after 30 min of exposure to Tyrode’s buffer (as a negative control, NS), TRAP, staphylococcal strains [clinical strains (SaB), S. condimenti (Sc) or S. aureus ATCC 43866 (ATCC43866)] or the A23187 apoptosis inducer ( as a positive control), with ASA, fluvastatin or water (as a control vehicle). ( a ) The data (mean ± SEM; n = 8) are expressed as AU. (b) The data (mean ± SEM; n = 8) are expressed as the % expression ( c ) The data (mean ± SEM; n = 9–27 experiments) are expressed as microparticles/µl. The data (mean ± SEM; n = 6–15 experiments) are expressed as the fold increase in active caspase-3 expression. **p < 0.01; ***p < 0.001, ****p < 0.0001 (two-way ANOVA with repeated measures and the Bonferroni post hoc test; ASA or fluvastatin vs . control vehicle).

Article Snippet: In brief, after stimulation, platelets were resuspended in Annexin Binding Buffer (MabTag, Oldenburg, Germany) for immunostaining.

Techniques: Binding Assay, Concentration Assay, Expressing, Flow Cytometry, Negative Control, Positive Control, Control

Journal: Nature Chemical Biology

Article Title: Menin-MLL Inhibitors Reverse Oncogenic Activity of MLL Fusion Proteins in Leukemia

doi: 10.1038/nchembio.773

Figure Lengend Snippet: (a) MTT cell viability assay in the MLL leukemia cells KOPN-8 and MV4;11 induced by MI-2, MI-3 and MI-nc after 72h treatment. Non-MLL leukemia cell line ME-1 is shown for comparison. Data represent mean values for four samples ± s.d. Experiment was performed three times. (b) Apoptosis and cell death induced by MI-2, MI-3 and MI-nc in MV4;11 cells as detected by flow cytometry using AnnexinV/propidium iodide (PI) staining. Data represent mean values for triplicates ± s.d. (c) Selected histograms from cell cycle analysis performed by FACS after PI staining in MV4;11 cells treated with DMSO, MI-2 or MI-nc. (d) Dose-dependent effect of MI-2 on cell cycle progression measured by FACS in MV4;11 cells after PI staining, with MI-nc as a negative control. Data represent mean values for triplicates ± s.d. (e) Wright-Giemsa stained cytospins on THP-1 and MV4;11 cells after 10 days of treatment with DMSO, MI-2 or MI-nc. (f) Detection of CD11b expression in THP-1 cells assessed by flow cytometry after 6 days of treatment with DMSO, MI-2 or MI-nc. Data represent mean values for triplicates ± s.d. (g) Expression of the HOXA9 and MEIS1 genes normalized to 18S rRNA determined by qRT-PCR in THP-1 cells treated for 6 days with MI-2 and MI-3. Data represent mean values for duplicates ± s.d. Experiment was performed three times.

Article Snippet: After incubation, 1.5×10 5 cells were harvested and resuspended in 100 μl 1× Annexin V binding buffer from the Annexin V-FITC Apoptosis kit (BD Biosciences Pharmingen), incubated with 4 μl of AnnexinV-FITC and 6 μl of Propidium iodide (Sigma-Aldrich) at room temperature in the dark for 10 minutes and analyzed by flow cytometry on a LSR II instrument.

Techniques: Viability Assay, Flow Cytometry, Staining, Cell Cycle Assay, Negative Control, Expressing, Quantitative RT-PCR