Journal: Human Mutation

Article Title: PLSCR3 Deficiency Triggers mtDNA‐Driven cGAS‐STING Activation to Potentiate Antitumor Immunity in Colorectal Cancer

doi: 10.1155/humu/8545428

Figure Lengend Snippet: PLSCR3 deficiency activates mtDNA‐associated cGAS‐STING signaling. (A) 2 ′ ‐3 ′ ‐cGAMP quantification by ELISA in PLSCR3 knockdown (KD_PLSCR3) HT29 cells and normal controls (NCs) treated with Scramble or dideoxycytidine (DDC) ( p < 0.0001; ns = not significant; n = 3). (B, C) KD_PLSCR3 cells exhibited an elevation in IFN β and CXCL10 production measured by ELISA compared to NC ( p < 0.0001, n = 3). CRISPR‐mediated STING knockout (STING sg) completely abrogated these effects. (D) Western blot analysis showed upregulation of phosphorylated STING and innate immune effectors (IFIH1, ISG60, and ISG15 protein) in STING‐competent KD_PLSCR3 cells relative to GAPDH loading controls. (E) Normalized mRNA level quantification by qPCR demonstrated significant induction of interferon‐stimulated genes (ISGs) in STING‐competent KD_PLSCR3 cells compared with the other groups. Data in panels A–C and E are presented as mean ± SD. Statistical significance was determined using one‐way ANOVA followed by Tukey′s multiple‐comparisons post hoc test for multiple‐group comparisons.

Article Snippet: STING agonist (Cat. No. HY‐103665, CAS No.: 2138299‐29‐1) and DDC (Cat. No. HY‐17392, CAS No.: 7481‐89‐2) were purchased from MedChemExpress (Shanghai, China).

Techniques: Enzyme-linked Immunosorbent Assay, Knockdown, CRISPR, Knock-Out, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: STING-STAT1-ZBP1 axis orchestrates PANoptotic signaling in ischemia-reperfusion induced acute kidney injury

doi: 10.1186/s12964-026-02830-2

Figure Lengend Snippet: ZBP1 deficiency alleviates PANoptosis induced by STING agonism. A ZBP1 mRNA levels (fold change) in primary tubular epithelial cells (PRTCs) treated with diABZI (STING agonist, 10 µM) for 6, 12, and 24 h (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. B ZBP1 mRNA levels (fold change) PRTCs treated with DMSO or diABZI in STING+/+-PRTCs and STING-/-- PRTCs (n = 3). *P < 0.05 ，**P < 0.01，***P <0.001. C Western blots and quantification of STING and ZBP1 protein PRTCs treated with DMSO or diABZI in STING+/+-PRTCs and STING-/-- PRTCs (n = 3). *P < 0.05 ，**P < 0.01，***P <0.001. D Immunofluorescence of ZBP1 (red), STING (green), and DAPI (blue) in PRTCs treated with diABZI (scale bars: 40 μm), The figure on the right is an enlarged view of the area within the white dashed box in the left one. E Inflammation markers: TNFα, IL1β mRNA levels and Cell viability assay with LDH release and Cell Viability Assay in ZBP1+/+-PRTCs and ZBP1-/--PRTCs treated with DMSO or diABZI (n = 4). *P < 0.05 ，**P < 0.01，***P <0.001. F Western blot analysis of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) in ZBP1+/+-PRTCs and ZBP1-/--PRTCs treated with DMSO or diABZI (n=3; α-Tubulin loading control). Red separator lines indicate different experimental conditions. G Flow cytometry quantification of cell death counts (PI+, V610) in ZBP1+/+-PRTCs and ZBP1-/--PRTCs treated with DMSO or diABZI (n=3)

Article Snippet: Primary cells were treated with diABZI (medchemexpress-HY-112921A), Cocl2 (sigma-232696), Fludarabine (medchemexpress-HY-B0069), Anifrolumab (medchemexpress-HY- P99168 ), IFN-α (medchemexpress-HY-P7023), Necrostatin-1 (medchemexpress-HY-15760), Z-VAD-FMK (medchemexpress-HY-16658B), MCC950 (medchemexpress-HY-12815), 2’3’-cGAMP (c-GAMP: tlrl-nacga23-02) for 24–48 h.

Techniques: Western Blot, Immunofluorescence, Viability Assay, Control, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: STING-STAT1-ZBP1 axis orchestrates PANoptotic signaling in ischemia-reperfusion induced acute kidney injury

doi: 10.1186/s12964-026-02830-2

Figure Lengend Snippet: STING induces ZBP1 activation in a STAT1-dependent manner. A Immunofluorescence of STAT1 (red), STING (green), and DAPI (blue) in renal cortices from STINGflox/flox-Sham, STINGflox/flox-IR, STINGCKO-Sham, and STINGCKO -IR groups, with quantification showing STING-dependent STAT1 upregulation during injury (n=5, scale bars: 50 μm). B Western blot confirmation of STAT1 and p-STAT1 protein expression trends in vivo (n=3; α-Tubulin loading control). C Nucleocytoplasmic fractionation in primary renal tubular epithelial cells (PRTCs) treated with PBS, 2’3’cGAMP (c-GAMP, 2 μg/ml), diABZI (10 μM), or H/R, revealing STAT1 and p-STAT1 nuclear accumulation upon STING activation (Lamin B/α-Tubulin: compartment markers). D Immunofluorescence confirming diABZI-driven STAT1 nuclear translocation in PRTCs (STAT1: red; DAPI: blue) (scale bars: 20 μm). E Confocal immunofluorescence microscopy images of PRTCs treated with diABZI, stained for STAT1 (red), STING (green), and DAPI (blue) (scale bars: 20 μm); a magnified detail of the white-boxed area is shown in the inset. The panel shows fluorescence co-localization analysis using the Plot Profile function in ImageJ-Fiji. F Molecular docking (GRAMM/HDOCK) predicted contact sites between STING (violet) and STAT1 (celeste); a magnified detail of the yellow-boxed area is shown in the inset. The black arrows represent the predicted hydrogen bonding. G Co-immunoprecipitation (Co-IP) of STING and STAT1 from PRTCs using antibodies against STAT1 and STING. Input: whole-cell lysate; IgG: negative control. H WT (HA-STING-WT) or mutant STING (HA-STING-CTD) expression vectors were cotransfected with Flag tagged STAT1 into 293T cells, and cell lysis was used for IP with anti-HA, followed by Western blot detection of STING or STAT1. I The schematic diagram of truncated plasmid of STING

Article Snippet: Primary cells were treated with diABZI (medchemexpress-HY-112921A), Cocl2 (sigma-232696), Fludarabine (medchemexpress-HY-B0069), Anifrolumab (medchemexpress-HY- P99168 ), IFN-α (medchemexpress-HY-P7023), Necrostatin-1 (medchemexpress-HY-15760), Z-VAD-FMK (medchemexpress-HY-16658B), MCC950 (medchemexpress-HY-12815), 2’3’-cGAMP (c-GAMP: tlrl-nacga23-02) for 24–48 h.

Techniques: Activation Assay, Immunofluorescence, Western Blot, Expressing, In Vivo, Control, Fractionation, Translocation Assay, Microscopy, Staining, Fluorescence, Immunoprecipitation, Co-Immunoprecipitation Assay, Negative Control, Mutagenesis, Lysis, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: STING-STAT1-ZBP1 axis orchestrates PANoptotic signaling in ischemia-reperfusion induced acute kidney injury

doi: 10.1186/s12964-026-02830-2

Figure Lengend Snippet: STING Drives STAT1-Mediated Transcriptional Activation of ZBP1 to Promote PANoptosis. A Using CUT-Tag assays in HK-2 cells treated with diABZI, isolated DNA fragments bound to STAT1 and identified potential binding sites on the ZBP1 promoter via Jaspar website. B Exploration of STAT1-ZBP1 promoter binding in diABZI-treated HK-2 cells: ZBP1 promoter region amplified using Primer3Plus-designed primers, Cleavage Under Targets and Tagmentation with STAT1 antibody compared with IgG control (n=3). C Western blot confirmation of ZBP1 and p-STAT1 protein expression trends in PRTCs treated with DMSO, diABZI, Fludarabine and diABZI groups (n=3; α-Tubulin loading control). D - E Fludarabine (STAT1 inhibitor) suppresses diABZI-induced ZBP1 mRNA and protein (n=4 mRNA, n=3 protein) and inflammatory cytokines (TNFα, IL1β mRNA; n=4) in PRTCs (*P<0.05, **P<0.01, ***P<0.001). F Viability assessment by PI (red, dead cells)/Calcein-AM (green, live cells) staining in PRTCs treated with DMSO, diABZI or diABZI and Fludarabine (n=3, scale bars: 50 μm). G PRTCs were treated with DMSO (control), diABZI (STING agonist), or diABZI and Fludarabine. Flow cytometry quantification of cell death (PI⁺, V610). H Western blot analysis of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) in PRTCs treated with DMSO, diABZI or diABZI and Fludarabine (n=3, α-Tubulin loading control)

Article Snippet: Primary cells were treated with diABZI (medchemexpress-HY-112921A), Cocl2 (sigma-232696), Fludarabine (medchemexpress-HY-B0069), Anifrolumab (medchemexpress-HY- P99168 ), IFN-α (medchemexpress-HY-P7023), Necrostatin-1 (medchemexpress-HY-15760), Z-VAD-FMK (medchemexpress-HY-16658B), MCC950 (medchemexpress-HY-12815), 2’3’-cGAMP (c-GAMP: tlrl-nacga23-02) for 24–48 h.

Techniques: Activation Assay, Isolation, Binding Assay, Amplification, Control, Western Blot, Expressing, Staining, Flow Cytometry

Journal: Cell Communication and Signaling : CCS

Article Title: STING-STAT1-ZBP1 axis orchestrates PANoptotic signaling in ischemia-reperfusion induced acute kidney injury

doi: 10.1186/s12964-026-02830-2

Figure Lengend Snippet: STING and IFNAR Pathways Cooperatively Promote STAT1 Activation Drives PANoptosis in Renal Tubular Epithelial Cells . A - B Primary renal tubular epithelial cells (PRTCs) were treated with DMSO (control), diABZI (STING agonist), or diABZI and Anifrolumab (IFNAR inhibitor). Representative Western blot analysis and quantification of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) and ZBP1 in PRTCs (n=3 α-Tubulin loading control). Red separator lines indicate different experimental conditions. C Primary renal tubular epithelial cells (PRTCs) were treated with DMSO, diABZI, or diABZI and Anifrolumab. Flow cytometry quantification of cell death (PI⁺, V610). D - E PRTCs were treated with DMSO, diABZI, or diABZI (10 μM) and IFN-α (1 ng/ml). Western blot analysis and quantification of PANoptosis markers (pyroptosis: GSDMD, N-GSDMD; apoptosis: CAS3, c-CAS3, CAS7, c-CAS7; necroptosis: p-RIP1, p-MLKL) in each group (n=3; α-Tubulin loading control). Red separator lines indicate different experimental conditions. F Proposed mechanism of the STING-STAT1-ZBP1 axis in renal tubular epithelial cells promoting pan-apoptosis in IRI-AKI. Following IRI-AKI, STING interacts with and activates STAT1, facilitating its nuclear translocation. STAT1 binds to the promoter region of the ZBP1 gene, initiating its transcription. The upregulated ZBP1 subsequently triggers the activation of PANoptosis, which drives the progression of AKI

Article Snippet: Primary cells were treated with diABZI (medchemexpress-HY-112921A), Cocl2 (sigma-232696), Fludarabine (medchemexpress-HY-B0069), Anifrolumab (medchemexpress-HY- P99168 ), IFN-α (medchemexpress-HY-P7023), Necrostatin-1 (medchemexpress-HY-15760), Z-VAD-FMK (medchemexpress-HY-16658B), MCC950 (medchemexpress-HY-12815), 2’3’-cGAMP (c-GAMP: tlrl-nacga23-02) for 24–48 h.

Techniques: Activation Assay, Control, Western Blot, Flow Cytometry, Translocation Assay

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative Airyscan-processed confocal images of HeLa STING and HeLa STING cells with stable overexpression of mEGFP-LC3B (green) treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta) and STING (cyan). Scale bar = 20 and 2 µm (inset). Imaging was replicated in two independent experiments. ( B ) Representative immunoblots of indicated proteins detected lysates from HeLa STING WT and HeLa STING with stable overexpression of mEGFP-LC3B prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in 3 independent experiments. ( C ) Representative immunoblots of indicated proteins detected in HeLa STING cell lysates prepared after treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( D , E ) Representative spinning disk confocal images of FRT/TREX HeLa cells stably expressing FRT/TO-DD-Vx3-EGFP, BFP-P2A-STING, and mScarletI-LC3B at the 6-hour timepoint following treatment ( D ) and quantification of the percentage (%) of cells positive for Vx3-EGFP foci over time ( E ). Cells were incubated with 1 µg/mL Doxycycline and 500 nM Shield1 for 24 h prior to treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI, and imaging every 30 min for 12 h on a spinning disk confocal microscope. Scale bar = 50 µm. Quantification is from three wells analyzed in the same experiment. Imaging was replicated in two independent experiments.

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Over Expression, Immunostaining, Ubiquitin Proteomics, Imaging, Western Blot, Stable Transfection, Expressing, Incubation, Microscopy

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and ATG16L1KO cells prepared following treatment with 1 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. ( B ) Representative immunoblots of indicated proteins detected in lysates from WT THP1 cells prepared following treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( C , D ) Relative expression changes of indicated NFκB-related genes ( C ) and interferon-related genes ( D ) detected by quantitative RT-PCR in THP1 cells treated with DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A one-way ANOVA with Tukey’s multiple comparisons test was performed on 2 −ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 ( TNF p = 0.0123; TNFAIP3 p = 0.005; IL6 p = 0.0027; IFNB1 p = <0.0001; ISG15 p = 0.0351).

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Recombinant, Ubiquitin Proteomics, Sequencing, CRISPR, shRNA, Reverse Transcription, SYBR Green Assay, Control, Magnetic Beads, Negative Control, Cloning, Modification, Software

Journal: bioRxiv

Article Title: The cGAS-STING pathway regulates microglial chemotaxis in genome instability

doi: 10.1101/2023.08.25.554654

Figure Lengend Snippet: (A) Representative immunoblot of STING pathway activation in ATM KO C20 cells (clones #1 and #2). Loading control: Vinculin. Quantification of phosphorylated proteins is relative to respective total proteins and normalised to WT. (B) Relative mRNA levels of IFNB1 and IFIT2 in WT and ATM KO C20 cells treated with 1 µM STING agonist diABZI, or DMSO as control, for 5 h. C q values normalised to IPO8 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (C) Representative immunoblot of HMC3 cells treated with 10 nM ATM inhibitor AZD1390 (ATMi), or DMSO as control, for 1, 3, 6 or 9 days. Loading control: Vinculin. NS: nonspecific. Quantification of phosphorylated proteins is relative to respective total proteins and normalised to Day 1 DMSO control. (D) Representative images of IRF3 localisation in HMC3 cells stimulated for 2 h with 1 µM diABZI, or DMSO as control, or with 10 nM ATMi or DMSO for 6 days (diABZI and ATMi stimulation are part of separate experiments). Red: IRF3, cyan: DNA (DAPI). 20x magnification, scale bar = 25 µm. Yellow arrows indicate cells with nuclear IRF3. (E) Percentage of cells with nuclear translocation of IRF3 treated as in (D). Mean ± SD (n=3). Unpaired, two-tailed Student’s t -tests. (F) Relative IFNB1 mRNA levels in HMC3 cells treated with ATMi or DMSO for 6 days followed by 1 µM diABZI, or DMSO as a control, for 5 h. C q values normalised to RPS13 and DMSO-treated cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant.

Article Snippet: To induce ATM activation, cells were treated with 1 μM camptothecin (CPT; Cayman Chemical Company) for 1 h. The STING agonist diABZi (Selleck Chemicals) was used at 1 μM.

Techniques: Western Blot, Activation Assay, Clone Assay, Control, Comparison, Translocation Assay, Two Tailed Test

Journal: bioRxiv

Article Title: The cGAS-STING pathway regulates microglial chemotaxis in genome instability

doi: 10.1101/2023.08.25.554654

Figure Lengend Snippet: (A) Relative mRNA levels of CCL5 and CXCL10 in WT and ATM KO C20 cells and upon treatment with 1 µM diABZI, or DMSO as control, for 5 h. C q values normalised to IPO8 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (B) Schematic of experimental workflow. WT and STING1 KO HMC3 cells were treated with 10 nM ATMi, or DMSO as a control, for 6 days. mRNA levels were measured by RT-qPCR. Secretion was measured 24 h after media change to low FBS supplemented with 1 µM diABZI, or DMSO as a control. (C) Relative mRNA levels of CCL5 and CXCL10 from cells treated as in (B). C q values normalised to RPS13 and DMSO-treated WT cells. Mean log 2 fold change ± SD (n=3). Two-way ANOVA with Tukey’s post-hoc comparison test. (D) CXCL10 concentration in cell culture supernatants. Mean ± SD (n=3). Dashed line indicates lower limit of detection (LLOD) = 0.12 pg/mL. Nd: not detected. One-way ANOVA with Tukey’s post-hoc comparison test in WT cells. (E) Representative immunoblot of cerebellar homogenates from individuals with A-T and matched individuals with no known diseases of the CNS as controls (Ctrl). Loading control: GAPDH. NS = nonspecific. (F) Relative mRNA levels of STING1 , CGAS , CCL5, and CXCL10 as in (E). C q values normalised to RPS13 . Mean 2 -δCT ± SD shown (n=3). Paired t -test. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001; ns = not significant.

Article Snippet: To induce ATM activation, cells were treated with 1 μM camptothecin (CPT; Cayman Chemical Company) for 1 h. The STING agonist diABZi (Selleck Chemicals) was used at 1 μM.

Techniques: Control, Comparison, Quantitative RT-PCR, Concentration Assay, Cell Culture, Western Blot