Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: ABPP proteomic analysis confirms VDAC1 as target protein of SEI. (A) Chemical structures of SEI and SEI probe (SEI‐P). (B) Cell viability of iBMDM cells treated with SEI or SEI‐P. (C) Release of inflammatory cytokine IL‐1β and (D) cell viability in TNF‐α plus IFN‐γ‐induced iBMDM cells. (E) Cellular imaging of SEI‐P with different exposure times in iBMDM cells. (F) Dose‐dependent labeling of proteins by SEI‐P in iBMDM cells. (G) Competition between SEI and SEI‐P for protein binding in situ (red star: 35 kD). (H) Chemical proteomics analysis workflow for identifying potential targets of SEI, created using Figdraw. (I) Volcano plot of proteins identified in the ABPP method. The graph displayed the log 2 FC of the competition group (100 µ m SEI + 50 µ m SEI‐P) versus SEI‐P (50 µ m ) ( x ‐axis) against the −log 10 ( p‐ value) ( y ‐axis). Among these, points with p < 0.05 and log 2 FC < −1 (blue) were selected as target protein candidates. Values were expressed as mean ± SD ( n = 3).

Article Snippet: Senkyunolide I (HY‐N0745), Erastin (HY‐15763), H‐151 (HY‐112693), TNF‐α (HY‐P7058 or HY‐P7090), IFN‐γ (HY‐P7025 or HY‐P7071), Lipopolysaccharides (LPS, HY‐D1056), Adenosine 5'‐triphosphate (ATP, HY‐B2176) were purchased from MedChem Express.

Techniques: Imaging, Labeling, Protein Binding, In Situ

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: SEI directly interacts with VDAC1 to inhibit its oligomerization. (A) The interaction between SEI and VDAC1 was detected using molecular docking. (B) Lysates from iBMDM cells were incubated with or without SEI (100 µ m ) for 24 h. Different concentrations of pronase E were added for 20 min, and VDAC1 content was analyzed using WB analysis. (C) Lysates from iBMDM cells were incubated with SEI at the indicated concentrations for 24 h, with a final concentration of 0.01% pronase E added for 20 min. The level of VDAC1 was assessed through WB analysis. (D) iBMDM cells were incubated with SEI (100 µ m ) for 24 h. These samples were then analyzed using CETSA. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01 versus SEI group. (E) MST demonstrating a direct interaction between SEI and EGFP‐tagged VDAC1 in lysates from EGFP‐VDAC1 expressing HEK293T cells. (F) Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells, untreated or stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (G) The VDAC1‐OE plasmid was transfected into iBMDM cells, and cGAMP production was subsequently measured by ELISA. (H) iBMDM cells were transfected with VDAC1‐OE, and then the phosphorylation levels of STING and IRF3 were measured by WB. (I) The mRNA expression levels of CCL5 , CXCL10 , and ISG15 in iBMDM cells transfected with VDAC1‐OE were measured by qRT‐PCR. (J) qRT‐PCR analysis of cytoplasmic mtDNA ( mt‐Nd1 , D‐loop and mt‐Cytb ). Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Senkyunolide I (HY‐N0745), Erastin (HY‐15763), H‐151 (HY‐112693), TNF‐α (HY‐P7058 or HY‐P7090), IFN‐γ (HY‐P7025 or HY‐P7071), Lipopolysaccharides (LPS, HY‐D1056), Adenosine 5'‐triphosphate (ATP, HY‐B2176) were purchased from MedChem Express.

Techniques: Incubation, Concentration Assay, Expressing, Western Blot, Plasmid Preparation, Transfection, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Quantitative RT-PCR

Journal: Advanced Science

Article Title: Senkyunolide I Inhibits mtDNA‐cGAS‐STING Signaling in Macrophages via Targeting VDAC1 Oligomerization to Attenuate Ulcerative Colitis

doi: 10.1002/advs.77045

Figure Lengend Snippet: SEI directly binds to K12 on VDAC1. (A) The RMSD of the VDAC1 backbone was simulated for a range of 100 ns. (B) The RMSF values of all amino acid residues were simulated. (C) Radius of gyration of the apo (blue) and protein when bound to the ligand (red) for the 100 ns simulation. (D) DCCM analysis matrix of VDAC1 protein; the region in red indicates residue pairs in horizontal and vertical coordinates have positive correlation in movement patterns, while the region in blue indicates negative correlation. (E) DCCM analysis matrix of SEI‐VDAC1 complex. (F–H) Movement correlation of each residue with a negative correlation coefficient ranged from −0.6 to −0.8 of VDAC1 protein (F), ranged from −0.4 to −0.6 of VDAC1 protein (G), and ranged from −0.4 to −0.6 of SEI‐VDAC1 complex (H). (I) The total binding free energy was calculated, and a series of contribution components were analyzed. Data are presented as energy changes in units of kJ/mol. (J) Ten residues of the VDAC1‐SEI complex were selected and analyzed. Data were presented as energy changes in a unit of kJ/mol with different contributors indicated by colors. (K) Free energy landscape. (L) Sequence conservation analysis of VDAC1 protein using ESPript 3.0. (M) The iBMDM cells were transfected with K174, K12, and G172 mutation plasmids and then treated with DMSO or SEI (100 µ m ) for 1 h. The interaction between SEI and VDAC1 was detected using the CETSA assay. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 versus Ctrl group. (N) VDAC1 −/− iBMDM cells were transfected with Flag‐VDAC1(WT), Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). Immunoblotting analysis of VDAC1 cross‐linking in iBMDM cells stimulated with TNF‐α plus IFN‐γ, with or without addition of SEI (100 µ m ). (O) VDAC1 −/− iBMDM cells were transfected with an empty vector, Flag‐VDAC1‐WT, Flag‐VDAC1(K174A), Flag‐VDAC1(K12A), or Flag‐VDAC1(G172A). WB analyses of the quantity of p‐TBK1 and p‐IRF3 after stimulation with TNF‐α plus IFN‐γ and treatment with SEI (100 µ m ) or left untreated (control) for 24 h. Values were expressed as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Senkyunolide I (HY‐N0745), Erastin (HY‐15763), H‐151 (HY‐112693), TNF‐α (HY‐P7058 or HY‐P7090), IFN‐γ (HY‐P7025 or HY‐P7071), Lipopolysaccharides (LPS, HY‐D1056), Adenosine 5'‐triphosphate (ATP, HY‐B2176) were purchased from MedChem Express.

Techniques: Residue, Binding Assay, Sequencing, Transfection, Mutagenesis, Western Blot, Plasmid Preparation, Control

Journal: Bioactive Materials

Article Title: Splenic dendritic cell-targeting mRNA transfection of H-type ionizable lipid-based LNPs for enhancing tumor immunotherapy

doi: 10.1016/j.bioactmat.2026.02.018

Figure Lengend Snippet: In vivo DC maturation and T cell activation in C57BL/6J mice induced by mOVA/H 18 NPs through intravenous injection. C57BL/6J mice were vaccinated with different formulations on Day 0 and Day 5. On Day 10, the spleens of mice were collected and analyzed by flow cytometry. Quantification analysis of (A) CD80 + CD86 + DCs and (B) CD40 + DCs in the spleen. Quantification analysis of (C) CD3 + CD4 + T cells and (D) CD3 + CD8 + T cells in the spleen. (E) Quantification analysis and (F) representative flow cytometry contour plots of OVA-specific CD8 + T cells among all cell populations in the spleen. (G) Quantification analysis and (H) representative flow cytometry contour plots of IFN-γ + CD8 + T cells among all cell populations in the spleen. (I) Quantification results and (J) representative images of IFN- γ -secreting immune cells in the spleen of mice analyzed by enzyme-linked immunospot (ELISpot) assay. Data were shown as mean ± SD (n = 3).

Article Snippet: Quantification analysis of (C) CD3 + CD4 + T cells and (D) CD3 + CD8 + T cells in the spleen. (E) Quantification analysis and (F) representative flow cytometry contour plots of OVA-specific CD8 + T cells among all cell populations in the spleen. (G) Quantification analysis and (H) representative flow cytometry contour plots of IFN-γ + CD8 + T cells among all cell populations in the spleen. (I) Quantification results and (J) representative images of IFN- γ -secreting immune cells in the spleen of mice analyzed by enzyme-linked immunospot (ELISpot) assay.

Techniques: In Vivo, Activation Assay, Injection, Flow Cytometry, Enzyme-linked Immunospot

Journal: iScience

Article Title: Distinct CD8 + T cell types associated with COVID-19 severity in unvaccinated HLA-A2 + patients

doi: 10.1016/j.isci.2026.115880

Figure Lengend Snippet: Information on 11 selected HLA-A2-binding 9-mer peptides derived from the SARS-CoV-2 genome (A) Workflow of HLA-A2-binding peptide prediction with NETMHCpan4.1 and IFN-γ ELISpot assay of PBMCs from 24 patients with COVID-19 using 31 predicted peptides ( and ). (B) Homology of 11 selected peptides among SARS-CoV-2 variants and other strains (as indicated). “YES” indicates that more than 90% of epitope sequences matched within 200 selected peptide sequences from the database . Otherwise, the % of matched sequences is shown. “NO” indicates no matched sequences. (C) Information on the SARS-CoV-2 genome and mapping of 11 selected peptides on its genome.

Article Snippet: anti-human-IFN-γ antibody , MABTECH , Cat# 3420-3-250; RRID: AB_907283.

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunospot

Journal: iScience

Article Title: Distinct CD8 + T cell types associated with COVID-19 severity in unvaccinated HLA-A2 + patients

doi: 10.1016/j.isci.2026.115880

Figure Lengend Snippet: IFN-γ ELISpot of CD8 + T cells in PBMCs from HLA-A2 + patients with COVID-19 using selected SARS-CoV-2 peptides (A) Cross-sectional ELISpot assay of HLA-A2-restricted CD8 + T cell responses against 11 selected peptides in PBMCs from 26 patients with mild, 8 with moderate, and 8 with severe COVID-19, respectively. (B) Log10-transformed mean spot-forming cell (SFC) counts (log10[Mean SFC +1]) per patient across 11 peptides in patients with mild, moderate, and severe COVID-19. Each point represents the mean response for one patient across 11 peptides ( n = 42 patients). A +1 pseudocount is added before the log10 transformation to accommodate zero values. Statistical comparisons were performed using a negative binomial mixed-effects model with Holm-Bonferroni correction for multiple comparisons. NS: not significant. (C) Longitudinal ELISpot assay of HLA-A2-restricted CD8 + T cell responses against selected peptides in PBMCs from patients with mild and moderate COVID-19 (as indicated).

Article Snippet: anti-human-IFN-γ antibody , MABTECH , Cat# 3420-3-250; RRID: AB_907283.

Techniques: Enzyme-linked Immunospot, Transformation Assay