cleaved c  (Quidel)

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: Assessment of survival, lung pathology, STING palmitoylation, and inflammatory markers in sepsis-induced acute lung injury. The cecal ligation and puncture (CLP) model was utilized to create sepsis-related acute lung injury (ALI). Samples were harvested for analysis 72 h after CLP. The NLRP3/AIM2-IN-3 group received an intravenous injection through the tail vein immediately after CLP induction at a dose of 10 mg/kg. ( A ) Survival rates were compared using Kaplan-Meier survival curves among Sham-operated mice, CLP-induced sepsis mice, and CLP-induced sepsis mice treated with the NLRP3/AIM2 inhibitor NLRP3/AIM2-IN-3 (CLP + NLRP3/AIM2-IN-3). ( B ) Lung tissues from Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups were subjected to hematoxylin and eosin (HE) staining for assessment of pathological changes (scale bar = 100 μm). ( C ) Lung injury was quantitatively assessed based on histological criteria, providing injury scores for Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. ( D ) STING palmitoylation levels in lung tissues were measured using the Acyl-resin-assisted capture (Acyl-RAC) assay in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. ( E ) Lung tissue sections underwent immunohistochemistry (IHC) staining to assess the expression of ZDHHC21, cleaved Caspase-1 (c-Caspase-1), and the cleaved form of Gasdermin D (GSDMD-N) in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups (scale bar = 100 μm). ( F ) Protein levels of ZDHHC21,TBK1, p-TBK1, IRF3, p-IRF3, c-Caspase-1, and GSDMD-N were quantified in lung tissues from Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups using Western blotting. ( G ) Levels of the inflammatory cytokines IL-18 and IL-1β in lung tissues were measured by enzyme-linked immunosorbent assay (ELISA) in Sham, CLP, and CLP + NLRP3/AIM2-IN-3 groups. Data are presented as mean ± SD. Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test. n = 8 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: Ligation, Injection, Staining, Immunohistochemistry, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: Evaluation of cell viability, pyroptosis, STING palmitoylation, and inflammatory markers in an in vitro sepsis-induced acute lung injury model. MLE-12 cells were stimulated with LPS (1 µg/mL) to induce a sepsis-induced ALI cell model, with the control group receiving an equivalent volume of PBS. All assays were performed 24 h after LPS stimulation. The NLRP3/AIM2-IN-3 group was treated with 10 µM of NLRP3/AIM2-IN-3 1 h prior to LPS stimulation. ( A ) The CCK-8 assay was conducted to assess the cell viability in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( B ) Proportions of PI-positive and Caspase-1-positive cells were analyzed by flow cytometry in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( C ) STING palmitoylation levels were assessed using the Acyl-resin-assisted capture (Acyl-RAC) assay in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( D ) ZDHHC21, TBK1, p-TBK1, IRF3, p-IRF3, cleaved Caspase-1 (c-Caspase-1), and GSDMD-N protein expression were determined by Western blot analysis in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. ( E ) IL-1β and IL-18 concentrations in the cell culture supernatant were measured by ELISA in control, LPS, and LPS + NLRP3/AIM2-IN-3 treated MLE-12 cells. Data are presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s post hoc test. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: In Vitro, Control, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: ZDHHC21 knockdown inhibits STING palmitoylation and pyroptosis in LPS-treated MLE-12 cells. ( A ) Western blot to verify successful knockdown of ZDHHC21 in control and shRNA-treated MLE-12 cells. ( B ) Western blot analysis of ZDHHC21 expression in control, LPS, LPS + shNC, and LPS + shZDHHC21 groups. ( C ) Cell viability was assessed using the CCK-8 assay across different treatment groups. ( D ) Flow cytometry analysis of Caspase-1/PI positive rates in each group. ( E ) Acyl-RAC assay to evaluate STING palmitoylation levels in control, LPS, LPS + shNC, and LPS + shZDHHC21 treated cells. ( F ) Western blot detection of cleaved Caspase-1 (c-Caspase-1), TBK1, p-TBK1, IRF3, p-IRF3, and GSDMD-N expression levels. ( G ) ELISA measurements of IL-18 and IL-1β levels in cell culture supernatant. Data are presented as mean ± SD. One-way ANOVA was employed to determine statistical significance, with Tukey’s post hoc test applied afterward. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: Knockdown, Western Blot, Control, shRNA, Expressing, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: AUF1 overexpression inhibits ETS2/ZDHHC21-mediated STING palmitoylation and pyroptosis in sepsis-induced ALI models. ( A ) Western blotting was utilized to measure AUF1 levels in lung tissues from Sham and CLP-induced sepsis mice. ( B ) Analysis of AUF1 protein levels in control and LPS-treated MLE-12 cells was carried out using Western blot. ( C ) Western blot analysis showing the expression levels of AUF1, ETS2, and ZDHHC21 in MLE-12 cells following AUF1 overexpression. NC: negative control; AUF1: AUF1 overexpression group. ( D ) Western blot analysis of AUF1, ETS2, and ZDHHC21 expression in MLE-12 cells under different conditions, including control, LPS, LPS + NC, and LPS + AUF1 overexpression. ( E ) Cell viability was assessed using the CCK-8 assay across different treatment groups. ( F ) Flow cytometry analysis of Caspase-1/PI-positive rates to evaluate pyroptosis levels. ( G ) Acyl-RAC assay to evaluate STING palmitoylation levels in different groups. ( H ) Western blot analysis of TBK1, p-TBK1, IRF3, p-IRF3, cleaved Caspase-1 (c-Caspase-1), and GSDMD-N expression levels. ( I ) ELISA measurements of IL-1β and IL-18 concentrations in cell culture supernatant. Data are presented as mean ± SD. Statistical significance was evaluated through one-way ANOVA with subsequent analysis using Tukey’s post hoc test, as well as two-tailed Student’s t-test for comparisons between two groups. n = 8 for A, n = 3 for B-I. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: Over Expression, Western Blot, Control, Expressing, Negative Control, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Two Tailed Test

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: AUF1 inhibits STING palmitoylation-mediated pyroptosis through the ETS2/ZDHHC21 axis in sepsis-induced ALI. ( A ) Western blot analysis of AUF1, ETS2, and ZDHHC21 protein expression in MLE-12 cells overexpressing AUF1, ETS2, or both. NC: negative control; AUF1: AUF1 overexpression group; ETS2: ETS2 overexpression group; AUF1 + ETS2: AUF1 and ETS2 both overexpression group. ( B ) Western blot analysis of AUF1, ETS2, and ZDHHC21 protein levels in MLE-12 cells treated with LPS following overexpression of AUF1, ETS2, or both. ( C ) CCK-8 assay measuring cell viability across different treatment groups following LPS stimulation. ( D ) Flow cytometry analysis of Caspase-1 and PI-positive rates to evaluate pyroptosis levels in each treatment group following LPS stimulation. ( E ) Acyl-RAC assay assessing STING palmitoylation levels in various treatment groups following LPS stimulation. ( F ) Western blot analysis of TBK1, p-TBK1, IRF3, p-IRF3, cleaved Caspase-1 (c-Caspase-1), and GSDMD-N in each treatment group following LPS stimulation. ( G ) ELISA measurements of pro-inflammatory cytokines IL-1β and IL-18 in cell culture supernatants across different treatment groups following LPS stimulation. Data are presented as mean ± SD. One-way ANOVA was performed to establish statistical significance, followed by Tukey’s post hoc test for detailed comparison. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: Western Blot, Expressing, Negative Control, Over Expression, CCK-8 Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Cell Culture, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Sepsis-induced acute lung injury: AUF1 regulates pyroptosis via ETS2/ZDHHC21-mediated STING palmitoylation

doi: 10.1007/s00018-025-06038-4

Figure Lengend Snippet: Molecular mechanism of AUF1-regulated pyroptosis via ETS2/ZDHHC21/STING pathway in ALI. In sepsis-induced ALI, AUF1 expression is downregulated in lung epithelial cells, resulting in reduced decay of ETS2 mRNA and subsequent upregulation of ETS2. Elevated ETS2 directly activates the transcription of ZDHHC21, a palmitoyltransferase, leading to increased STING palmitoylation. Activated STING signaling promotes the phosphorylation of TBK1 and IRF3, ultimately enhancing pyroptosis via upregulation of c-Caspase-1, GSDMD-N, IL-18, and IL-1β. Overexpression of AUF1 promotes the decay of ETS2 mRNA, thereby suppressing the ETS2/ZDHHC21/STING axis, reducing STING palmitoylation and downstream pyroptosis, and alleviating lung injury. This pathway highlights AUF1 as a potential therapeutic target for sepsis-induced ALI

Article Snippet: Sections were incubated overnight at 4 °C with primary antibodies against ZDHHC21 (1:200, Invitrogen, PA5-25096), c-Caspase-1 (1:200, HY- P80622 ; MedChemExpress), and GSDMD-N (1:200, Invitrogen, PA5-116815).

Techniques: Expressing, Phospho-proteomics, Over Expression

Journal: Molecular Therapy Oncology

Article Title: Trabectedin decreases myeloid resistance to improve the efficacy of anti-PD1 immunotherapy and delay glioma malignant progression

doi: 10.1016/j.omton.2026.201147

Figure Lengend Snippet: Combination trabectedin and anti-PD1 ICB prolongs survival of glioma-bearing mice (A) Schematic depicting the treatment regimen for the Ntv-a x XFM-luc RCAS murine glioma progression model. Four groups of mice were treated with vehicle, trabectedin (2 doses, 1 week apart, starting at week 4), anti-PD-1 immune checkpoint blockade (ICB; 3 doses, 3 days apart, starting at week 3.5), or a combination of trabectedin and anti-PD-1 ICB. (B) Kaplan-Meier survival analysis of the four treatment groups. Vehicle ( n = 12), anti-PD1 ( n = 8), trabectedin ( n = 13), and trabectedin + anti-PD1 ( n = 12) had median survivals of 39, 43, 43, and 53 days, respectively. Significance was determined using the log-rank (Mantel-Cox) test ( p < 0.0001∗∗∗∗, p < 0.0002 ∗∗∗, p < 0.0021∗∗, p < 0.0332∗). (C) Representative images of cleaved caspase-3 (c-cas3) immunohistochemical (IHC) staining of tumors isolated from glioma-bearing mice 3 days post treatment 2 (3DPT2) with vehicle, trabectedin, or trabectedin + anti-PD1 combination. (D) Quantification of c-cas3 IHC staining. Positive pixels were calculated by sampling 5 equal-sized regions within each tumor core and comparing the number of positive pixels per region ( n = 2–5 mice per treatment group). Significance was determined using a paired t test with Welch’s correction ( p < 0.0005∗∗∗∗, p < 0.005 ∗∗∗, p < 0.005∗∗, p < 0.05∗). Error bars show SD.

Article Snippet: Slides were washed in TBST and stained with c-cas3 (R&D Systems MAB835, Minneapolis, MN) at a 1:100 dilution for 2 h at room temperature.

Techniques: Immunohistochemical staining, Immunohistochemistry, Isolation, Sampling

Journal: International Journal of Biological Sciences

Article Title: CCL24 recruits CCR3 + TAMs to promote immunosuppression via YAP1 activation and serves as a therapeutic target for Gracillin in colorectal cancer

doi: 10.7150/ijbs.127341

Figure Lengend Snippet: CCL24 blockade reduces CRC incidence and progression in vivo . A, Correlation analysis of CCL24 expression with T cell dysfunction and TAM-related marker genes in the TCGA-COAD dataset. B, TMA analysis of CCL24 staining intensity in tumor and adjacent tissues from 84 CRC patients. C-D, qPCR and ELISA detection of CCL24 mRNA and secretion levels in colon epithelial cells (FHC) and CRC cell lines (HCT116, Lovo, SW480, MC38). E, a primary CRC model was established in C57BL/6 mice through AOM/DSS induction, followed by tail vein injection of anti-CCL24 (ACl24, 10 μg/kg/week) after 8 weeks for CCL24 blockade. F, Gross images of mouse colons with primary tumors. G, Tumor number and diameter in mouse colons. H, IHC detection of C-Cas-3 positive staining in tumor tissues. I, Flow cytometry analysis of Arg1 + Mrc1 + TAMs in mouse colon tissues. TMA includes 84 pairs of tissue samples. Cell experiments repeated 6-8 times. Animal experiments include 6-8 mice per group. Data are presented as bars and dots. p < 0.05 was considered statistically significant.

Article Snippet: IHC was performed using anti-Cleaved-Caspase-3 (C-Cas-3) and anti-CCR3 antibodies (Cell Signaling Technology), with secondary antibodies visualized using a DAB detection system (ZSGB-BIO).

Techniques: In Vivo, Expressing, Marker, Staining, Enzyme-linked Immunosorbent Assay, Injection, Flow Cytometry

Journal: International Journal of Biological Sciences

Article Title: CCL24 recruits CCR3 + TAMs to promote immunosuppression via YAP1 activation and serves as a therapeutic target for Gracillin in colorectal cancer

doi: 10.7150/ijbs.127341

Figure Lengend Snippet: CCL24 knockout in colon epithelium reduces AOM/DSS-induced tumorigenesis and enhances CD8 + T cell function in mice. A, CCL24 fl/fl and Villin-Cre mice were allowed to mate to generate mice with colon epithelium-specific CCL24 conditional knockout (C24 cko ), followed by AOM/DSS challenge to induce colorectal tumorigenesis. B, Gross images of mouse colons. C, Tumor number and diameter in mouse colons. D-F, Populations of total CD8 + T cells (D), TNFA + (E), and GZMB + (F) CD8 + T cells in mouse tumor tissues. G, Anti-CD8 antibody treatment for CD8 + T cell depletion in AOM/DSS-challenged C24 cko mice after 8 weeks. H, Gross images of mouse colons. I, Tumor number and diameter in mouse colons. J, IHC detection of C-Cas-3 staining in tumor tissues. Each group includes 6-8 mice. Data are presented as bars and dots. p < 0.05 was considered statistically significant.

Article Snippet: IHC was performed using anti-Cleaved-Caspase-3 (C-Cas-3) and anti-CCR3 antibodies (Cell Signaling Technology), with secondary antibodies visualized using a DAB detection system (ZSGB-BIO).

Techniques: Knock-Out, Cell Function Assay, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Ubiquitination of Rhomboid 5 Homolog 2 by Constitutive Photomorphogenic 1 Alleviates Hepatic Ischemia-reperfusion Injury by Regulating the Transforming Growth Factor-β Activating Kinase 1-C-Jun N-terminal Kinase/p38 Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101695

Figure Lengend Snippet: Overexpression of Rhbdf2 exacerbated H/R-induced apoptosis and inflammatory response, whereas downregulation of Rhbdf2 significantly alleviates H/R-induced liver injury. ( A ) Analysis of Rhbdf2 protein and mRNA expression levels in Rhbdf2 overexpressing cell lines (OE) and vector control cell lines (VT) by Western blot and qRT-PCR analyses (n = 3 per group). ( B ) Western blot analysis of the protein expression levels and quantification of Bax, Bcl2, and C-caspase 3 in the OE group and the VT group before and after H/R (n = 3 per group). ( C ) Flow cytometry analysis of apoptosis rate and statistical analysis of cells in the OE and VT groups after H/R (n = 3 per group). ( D ) qRT-PCR analysis of mRNA levels of IL-6, IL-1β, and TNF-α in the OE and VT groups after H/R (n = 3 per group). ( E ) Western blot analysis and quantification of the expression levels of NF-κB signaling pathway-related proteins in cells from the OE and VT groups before and after H/R (n = 3 per group). ( F ) Western blot analysis and qRT-PCR analysis to determine the protein and mRNA expression levels of Rhbdf2 respectively in Rhbdf2 knockdown cells (SH1, 2, 3) and control cells (NC) (n = 3 per group). ( G ) Western blot analysis of the protein expression levels of Bax, Bcl2, and C-caspase3 in the cells of the SH1 and NC groups before and after H/R, and the results of statistical analysis (n = 3 per group). ( H ) Flow cytometry analysis of apoptosis rate of SH1 cells and NC cells before and after H/R, and statistical analysis (n = 3 per group). ( I ) qRT-PCR analysis of mRNA expression levels of inflammatory cytokines IL-6 , IL-1β, and TNF-α in SH1 and NC cells before and after H/R (n = 3 per group). ( J ) The expression levels and statistical analysis of NF-κB signaling pathway-related proteins in SH1 and NC cells before and after H/R (n = 3 per group). Using β-Actin as loading control; data are shown as the mean ± SD; ns indicates no statistical difference compared with control; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 indicate a statistical difference compared with control.

Article Snippet: C-caspase3 , CST , 9661T.

Techniques: Over Expression, Expressing, Plasmid Preparation, Control, Western Blot, Quantitative RT-PCR, Flow Cytometry, Knockdown

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Ubiquitination of Rhomboid 5 Homolog 2 by Constitutive Photomorphogenic 1 Alleviates Hepatic Ischemia-reperfusion Injury by Regulating the Transforming Growth Factor-β Activating Kinase 1-C-Jun N-terminal Kinase/p38 Signaling Pathway

doi: 10.1016/j.jcmgh.2025.101695

Figure Lengend Snippet: Overexpression of Rhbdf2 inhibits the protective effect of Cop1. ( A ) Plasmids carrying the indicated genes (VT, Cop1, Rhbdf2, Cop1+Rhbdf2) were transfected into AML12, and apoptotic rate of each group was determined by flow cytometry after H/R and statistical analysis (n = 3 per group). ( B ) Western blot analysis and statistical analysis of the expression levels of Bax, Bcl2, and C-caspase3 proteins in the indicated groups after H/R (n = 3 per group). ( C ) RT-PCR analysis of mRNA levels of IL-6, IL-1β , and TNF-α in the indicated groups after H/R (n = 3 per group). ( D ) Western blot analysis and quantitative analysis of the expression levels of NF-κB signaling pathway-associated proteins in each group after H/R (n = 3 per group). ( E ) Western blot analysis and quantification of the expression levels of TAK1, JNK, and p38 proteins and their phosphorylated proteins in each group after H/R (n = 3 per group). Using β-Actin as loading control; data are shown as the mean ± SD; ns indicates no statistical difference compared with control; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 indicate a statistical difference compared with control.

Article Snippet: C-caspase3 , CST , 9661T.

Techniques: Over Expression, Transfection, Flow Cytometry, Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Control