Journal: bioRxiv

Article Title: Cell autonomous inflammation in VEXAS is mediated by cGAS-STING

doi: 10.64898/2026.05.26.727520

Figure Lengend Snippet: ( A ) Vacuole-containing VEXAS THP1 cells contain less mitochondria, as demonstrated by brightfield (BF) and single-plane confocal anti-TOM20 immunofluorescence images of UBA1 WT and UBA1 M41V THP1 monocytes treated with doxycycline for 10 d. Representative images of 2 biological replicates are shown. For more examples, see Fig. S16. Scale bar = 10 μm. ( B ) UBA1 M41V THP1 monocytes exhibit mitochondrial morphology abnormalities, indicative of impaired function. UBA1 WT and UBA1 M41V THP1 cells were treated with dox for 10 d and analyzed by transmission electron microscopy. Scale bar = 1 μm. Red arrowheads point to dilated cristae often seen in UBA1 M41V mitochondria (see also Movie S2) ( C ) UBA1 M41V THP1 monocytes exhibit a higher percentage of mitochondria with dilated cristae per cell as compared to UBA1 WT control cells. Graphs depict quantifications of mitochondrial morphology on TEM images of the experiment shown in panel b. n > 20 cells, error bar = s.e.m, * = p < 0.05, ** = p < 0.01, student’s t-test. ( D ) VEXAS THP1 cells exhibit elevated levels of cytoplasmic mitochondrial DNA, as revealed by qPCR analysis of whole cell or cytoplasmic fractions of UBA1 WT and UBA1 M41V THP1 monocytes. n = 3 biological replicates with 2 technical replicates for two different mitochondrial DNA probes (see methods), error bar = s.e.m, ** = p < 0.01, student’s t-test. ( E ) Schematic representation of experimental design to test whether mtDNA release into the cytosol activates cGAS-STING-based VEXAS inflammation. ( F ) Blocking mtDNA release into the cytosol using either a scavenger of reactive oxygen species (MitoTEMPO), an inhibitor of VDAC oligomerization (VBIT4), or an inhibitor of the mitochondrial permeability transition pore (CsA) dampens STING signaling and attenuates VEXAS inflammation in THP1 UBA1 M41V model cells. UBA1 WT and UBA1 M41V THP1 monocytes were treated with indicated inhibitors (10 μM MitoTEMPO, 10 μM VDAC, and 1 μM CsA) for 24h and subjected to immunoblot analysis using indicated antibodies. Graphs depict quantifications of p-STING/STING, p-STAT1/STAT1, or p-STAT3/STAT3 ratio normalized to DMSO-treated UBA1 WT THP1 monocytes. n = 3 biological replicates, error bar = s.e.m, * = p < 0.05, ** = p < 0.01, student’s t-test. ( G ) Blocking mtDNA release into the cytosol restores STING localization by preventing its trafficking to the Golgi, as evidenced by anti-STING and anti-GM130 immunofluorescence analysis of THP1 model monocytes treated as described in panel F. ( H ) Quantification of the percentage of Golgi-localized STING of the experiment depicted in panel g. n ≥ 150 cells across 10 fields, error bar = s.e.m, **** = p < 0.0001, one-way ANOVA. ( I ) Verification that treatment of THP1 VEXAS model cells with indicated inhibitors blocks mtDNA release into the cytosol, as revealed by qPCR analysis of cytoplasmic fractions of UBA1 WT and UBA1 M41V THP1 monocytes. n = 3 biological replicates, 2 technical replicates for two different mitochondrial DNA probes (see methods), error bar = s.e.m, **** = p < 0.0001, one-way ANOVA. (J) Scheme depicting the molecular mechanisms triggering STING activation in VEXAS myeloid cells. Mitochondria in VEXAS myeloid cells are defective and release mtDNA into the cytosol, causing activation of cGAS-STING signaling, which drives multi-cytokine inflammation.

Article Snippet: Cells were treated for specified time periods with the following: H-151 (1 μM or 10 μM; MedChemExpress, HY-112693), SN-011 (10 μM; MedChemExpress HY-145010), MitoTEMPO (10 μM; MedChemExpress, HY-112879), Cyclosporin A (1 μM; MedChemExpress HY-B0579), VBIT4 (10 μM; MedChemExpress, HY-129122), 4μ8c (100 μM; MedChemExpress, HY-19707), GSK2606414 (5 μM; MedChemExpress, HY-18072), Ceapin-A7 (6 μM; MedChemExpress, HY-108434), PKR-IN-C16 (0.6 μM; MedChemExpress, HY-13977A), GCN2-IN-1 (1 μM; MedChemExpress, HY-100877), CP26 (1 μM, 1.25 μM, 2 μM, and 5 μM; MedChemExpress, HY-49116), Eeyarestatin I (10 μM; MedChemExpress, HY-110078), TAK-243 (5 nM; MedChemExpress, HY-100487), cyclohexamide (50 μg/mL; Sigma Aldrich, C4859), BafA1 (1 μM or 10 μM; MedChemExpress, HY-100558), and NSC697923 (3 μM or 4 μM; MedChem Express, HY-13811).

Techniques: Immunofluorescence, Transmission Assay, Electron Microscopy, Control, Blocking Assay, Permeability, Western Blot, Activation Assay

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: Drp1-dependent VDAC1 oligomerization is required for P. gingivalis induced mitochondrial dysfunction. HAECs was pretreated with or without Mdivi-1 (50 μM) and then infected with P. gingivalis for 24 h (MOI = 100). (A, B) Representative immunofluorescence images and statistics analysis showing the co-location of VDAC1 (red) and p -Drp1 (green) in HAECs. Scale bars: 20 μm (original) and 2 μm (Zoom). (C) The interaction of p -Drp1 and VDAC1 was validated using Co-immunoprecipitation assays. ( n = 3). (D, E) Immunoblot of VDAC1 cross-linking in HAECs pretreated with Mdivi-1 (50 μM) or VBIT-4 (20 μM) and quantitative analysis of oligomers. ( n = 3). (F, G) Calcein, MitoSOX staining assay and quantitative bar chart. Scale bars = 20 μm. ( n = 3) (H, I) Tube formation assay and quantitative bar chart. Scale bars = 200 μm. ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. * P < 0.05. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Immunofluorescence, Immunoprecipitation, Western Blot, Staining, Tube Formation Assay

Journal: Journal of Oral Microbiology

Article Title: P. gingivalis induces endothelial dysfunction via mitochondrial fission dependent VDAC1-HK2 disassociation

doi: 10.1080/20002297.2026.2643035

Figure Lengend Snippet: P. gingivalis triggers HK2 dissociation from VDAC1. HAECs were pretreated with Mdivi-1 (50 μM) or VBIT-4 (20 μM), followed by infection with P. gingivalis for 24 h. (MOI = 100). (A, B) Representative immunofluorescence images and statistics analysis showing the co-location of VDAC1 (red) and HK2 (green) in HAECs. Scale bars: 20 μm (original) and 5 μm (Zoom). (C) The interaction of VDAC1 and HK2 was validated using Co-immunoprecipitation assays. ( n = 3). (D, E) Representative immunoblots and quantification of HK2 levels in cytosolic and mitochondrial fractions of HAECs ( n = 3). (F, G) Representative immunofluorescence images and statistics analysis showing the co-location of mitochondria (red) and HK2 (green) in HAECs. Scale bars: 20 μm (original) and 5 μm (Zoom). ( n = 3). Pg, P. gingivalis . All numbers ( n ) are biologically independent experiments. ns = not significant. * P < 0.05. ** P < 0.01. *** P < 0.001.

Article Snippet: Mdivi-1, the mPTP inhibitor cyclosporin A (CsA), and the VDAC1 oligomerization inhibitor VBIT-4 were purchased from TargetMol (USA).

Techniques: Infection, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Advanced Science

Article Title: CypD Dependent mPTP Opening Is Crucial for Oxidized Mitochondrial DNA Release in Ferroptosis

doi: 10.1002/advs.202502239

Figure Lengend Snippet: OGG1 inhibition promotes ferroptosis via an mPTP‐dependent mechanism. (A) NCI‐H1299 cells were treated with increased doses of RSL3 as indicated in the presence of TH5487 (5 µ m ) and Fer‐1 (2 µ m ) for 8 h; then cell survival rate was measured by Cell Titer Glo Viability Assay. (B) The morphology of NCI‐H1299 cells treated with RSL3 (0.5 µ m ) for 4 h following TH5487 (5 µ m ) and Fer‐1 (2 µ m ) pre‐treatment. Scale bar was 50 µm. (C) NCI‐H1299 were treated with 5 µ m erastin as indicated in the presence of TH5487 (5 µ m ) and Ferrostatin (Fer‐1, 2 µ m ) for 18 h, and the lipid peroxidation level was determined using C11‐BODIPY staining by flow cytometry. (D) Quantitative analysis of the fold change of lipid oxidation ratio in (C). (E) GPX4 siRNA were transfected to HT‐1080 cells, after 36 h, TH5487, SU0268 and Fer‐1 were added as indicated, then cells viability was detected using Cell Titer Glo. (F) NCI‐H1299 were treated with increased doses of erastin as indicated in the presence of TH5487 (5 µ m ), with or without VBIT‐4 (5 µ m ) pretreatment for 8 h, then cell survival rate was measured by Cell Titer Glo Viability Assay. (G) NCI‐H1299 cells were treated with increased doses of erastin as indicated in the presence of TH5487 (5 µ m ), with or without CsA (5 µ m ) pretreatment for 24 h, and then the cell survival rate was measured by Cell Titer Glo Viability Assay. (H) CypD were knocked out in NCI‐H1299 cells, then the cells were treated with increased doses of erastin with or without TH5487 (5 µ m ) pretreatment. Cell death was measured by Cell Titer Glo Viability Assay. (I, J) ANT2 (I) and ANT3(J) were knocked down by specific siRNA in NCI‐H1299 cells, then the cells were treated with increased doses of erastin with or without TH5487 (5 µ m ) pretreatment. Cell death was measured by Cell Titer Glo Viability Assay. The statistical significance between different groups (D and E) was analyzed by Two‐Way ANOVA (Prism; GraphPad).

Article Snippet: RSL3 (T3646), erastin (T1765), ML210 (T8375), FINO2 ( T60084 ), CsA (T0945), DFO (T124358), BSO (T5471), TH5487 (T8119), SU0268 (T9119), Fer‐1 (T6500), VBIT‐4 ( T13287 ), H‐151 (T5674) and RU.521 (T5486) were ordered from TargetMol; Arachidonic acid (HY‐109590), Ardenic acid (HY‐W013215), and NIM811 (HY‐P0025) was purchased from MedChemExpress; IKE (MC2024) was purchased from MeilunBio.

Techniques: Inhibition, Viability Assay, Staining, Flow Cytometry, Transfection