human cleaved gsdmd  (Cell Signaling Technology Inc)

Journal: iScience

Article Title: FOXE1 promotes the progression of pulp inflammation by activating PANoptosis in dental pulp cells

doi: 10.1016/j.isci.2026.115204

Figure Lengend Snippet: Mixed infection with S.m. and F.n. induced PANoptosis in DPCs (A–H) Dental pulp cells were infected with S.m. (MOI = 100), F.n. (MOI = 100), or a combination of S.m. (MOI = 100) and F.n. (MOI = 100) or treated with PBS (negative control, NC) for 8 h. (A) Dental pulp cells were treated with PBS, S.m. , F.n. , or a combination of S.m. and F.n. ( n = 5 experiments in each group), and cell death was quantified by measuring LDH release. (B) Dental pulp cells were treated with PBS, S.m. , F.n. , or a combination of S.m. and F.n . Treated DPCs were stained with PI, fixed, counterstained with DAPI, and visualized under a microscope. Scale bars, 100 μm. (C) Quantification of the proportions of PI-positive cells in B ( n = 6 experiments in each group). (D) Dental pulp cells were treated as indicated and analyzed by flow cytometry. (E) PANoptotic DPCs were quantified by determining the percentages of PI + /annexin V + cells ( n = 3 experiments in each group). (F) The protein levels of cleaved caspase-3 p17, cleaved GSDMD NT, and p -MLKL were assessed by Western blot. (G) Immunofluorescence staining of PANoptotic markers in infected DPCs, as visualized using a confocal microscope. Scale bars, 10 μm. (H) Quantification of PANoptotic cell proportions in DPCs treated with PBS, S.m. , F.n. , or a combination of S.m. and F.n. ( n = 8 samples in each group), corresponding to (G). Data are represented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by one-way ANOVA (A, C, and E) or by Kruskal-Wallis test (H).

Article Snippet: The following primary antibodies were used in this study: human and mouse vimentin (1:200, #ab92547, Abcam), human cleaved caspase-3 (1:100, #9664, CST), human cleaved GSDMD (1:100, #36425, CST), human p-MLKL (1:100, #PA5-105678, Invitrogen), mouse cleaved caspase-3 (1:100, #BF0711, Affinity), mouse cleaved GSDMD (1:100, #ab255603, Abcam), mouse p-MLKL (1:100, #ab196436, Abcam) and mouse FOXE1 (1:50, #55363-1-AP, Proteintech).

Techniques: Infection, Negative Control, Staining, Microscopy, Flow Cytometry, Western Blot, Immunofluorescence

Journal: iScience

Article Title: FOXE1 promotes the progression of pulp inflammation by activating PANoptosis in dental pulp cells

doi: 10.1016/j.isci.2026.115204

Figure Lengend Snippet: Identification and validation of FOXE1 as a key transcription factor that regulates PANoptosis in DPCs (A) Differentially expressed gene analysis was performed between the PANoptotic DPC cluster (cluster 1) and other DPC clusters (clusters 0, 2, 4, 5, and 6) and between cluster 1 and all remaining clusters. The candidate regulators were selected from the overlapping DEGs between these comparisons. (B) The top five candidate regulators were identified after comparison. (C) siRNA targeting FOXE1 (20 μM) was transfected into DPCs with Lipofectamine 2000 for 24 h. The knockdown efficiency was validated by qRT-PCR ( n = 3 experiments in each group) and Western blot. (D–H) Dental pulp cells were pretreated with siRNAs (20 μM) targeting candidate regulators for 24 h, followed by infection with S.m. (MOI = 100) and F.n. (MOI = 100) for 8 h. (D) Cell death was quantified by the LDH release assays ( n = 5 experiments in each group). (E, also see in ) Cell death was assessed by PI staining ( n = 4 experiments in each group). (F) Treated cells were analyzed by flow cytometry. (G) PANoptotic DPCs were quantified by determining the percentages of PI + /annexin V + cells ( n = 3 experiments in each group). (H) Western blot analysis was used to assess the expression of cleaved caspase-3 p17, cleaved GSDMD NT and p -MLKL. (I) Immunofluorescence staining of PANoptotic markers in si FOXE1 -and siCON-transfected DPCs. Scale bars, 10 μm. (J) Quantification of PANoptotic DPCs in I ( n = 5 samples in each group). Data are represented as mean ± SEM. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 by Student’s t test (C, G, and J) or by one-way ANOVA (D and E).

Article Snippet: The following primary antibodies were used in this study: human and mouse vimentin (1:200, #ab92547, Abcam), human cleaved caspase-3 (1:100, #9664, CST), human cleaved GSDMD (1:100, #36425, CST), human p-MLKL (1:100, #PA5-105678, Invitrogen), mouse cleaved caspase-3 (1:100, #BF0711, Affinity), mouse cleaved GSDMD (1:100, #ab255603, Abcam), mouse p-MLKL (1:100, #ab196436, Abcam) and mouse FOXE1 (1:50, #55363-1-AP, Proteintech).

Techniques: Biomarker Discovery, Comparison, Transfection, Knockdown, Quantitative RT-PCR, Western Blot, Infection, Staining, Flow Cytometry, Expressing, Immunofluorescence

Journal: Nature Immunology

Article Title: Human T H 17 cells engage gasdermin E pores to release IL-1α on NLRP3 inflammasome activation

doi: 10.1038/s41590-022-01386-w

Figure Lengend Snippet: a , Differential gene expression determined by transcriptome analysis of T H 17 cells treated as in Fig. ( n = 3 individual healthy blood donors). b , RT–qPCR analysis of anti-CD3 and anti-CD28 monoclonal antibody-stimulated, naive T cells in polarizing cytokine conditions ( n = 4, one-way ANOVA with Dunnett’s multiple-comparison test). c , Immunoblot analysis of cell lysates from T H 17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for different durations. The data represent three experiments. d , ELISA of cell culture supernatants from T H 17 cells with and without deletion of GSDME (left) or GSDMD (right) by CRISPR–Cas9 technology. Individual experiments were normalized to the first time point of analysis on day 2 ( n = 3 individual biological samples, two-way ANOVA with Bonferroni’s multiple-comparison test). e , Immunoblot analysis of cell lysates from T H 17 cells stimulated with anti-CD3 and anti-CD28 monoclonal antibodies for the indicated time points and of CD14 + monocytes stimulated for 24 h with LPS and 30 min with nigericin. Casp, Caspase. f , Lane view of electropherograms obtained with a Jess Simple Western System for cell lysates of T H 17 cells stimulated for 5 d as in e in the presence or absence of the indicated inhibitors. It is a representative experiment. g , Cumulative data of f (one-sample Student’s t -test). AUC, area under the curve. h , Luminex assay of the supernatants of T H 17 cells stimulated with plate-bound anti-CD3 (1 μg ml −1 , TR66) and phorbol-12,13-dibutyrate for 8 h on day 4 of culture ( n = 3 individual biological samples, two-tailed, paired Student’s t -test). i , ELISA of supernatants of T H 17 cells stimulated as in f . Each circle indicates an independent blood donor in h and i ( n = 4 individual biological samples; two-tailed, paired Student’s t -test).

Article Snippet: The following primary antibodies were used for immunoblotting: mouse anti-human caspase-8 (Cell Signaling Technology), rabbit anti-human caspase-1 (Cell Signaling Technology), rabbit anti-human IL-1α (Abcam), mouse anti-human glyceraldehyde-2-phosphate dehydrogenase (GAPDH; Merck Millipore), mouse anti-human β-actin (Cell Signaling Technology) and rabbit anti-human GSDME (Abcam), rabbit anti-human caspase-3 (Cell Signaling Technology), mouse anti-human caspase-8 (Cell Signaling Technology), rabbit anti-human GSDMD (Cell Signaling Technology), rabbit anti-human cleaved GSDMD (Cell Signaling Technology) and rabbit anti-NLRP3 (Cell Signaling Technology).

Techniques: Gene Expression, Quantitative RT-PCR, Comparison, Western Blot, Bioprocessing, Enzyme-linked Immunosorbent Assay, Cell Culture, CRISPR, Simple Western, Luminex, Two Tailed Test