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: Integrating complementary approaches reveals antigen-reactive CD4 + T cell states after SARS-CoV-2 vaccination

doi: 10.1016/j.isci.2026.116175

Figure Lengend Snippet: Identification of SARS-CoV-2-specific T cell responses by reverse phenotyping (A) CoVa-Adapt study design and sample collection scheme. For all donors, PBMCs were collected at day 0 (P0), 10 days after primary (P10), 10 and 210 days after secondary (S10, S210), and 10 and 189 days after tertiary (T10, T189) vaccination. For selected donors, PBMCs were additionally sampled 108 days after tertiary vaccination (T108, n = 7). Vaccination-induced T cell responses were characterized for most donors on a quantitative level by IFNγ ELISpot. Selected CoVa-Adapt donors were subjected to in-depth characterization using scRNAseq (reverse phenotyping and epitope-specific analyses) followed by TCR functional testing. (B–G) scRNAseq data from the reverse phenotyping dataset. For reverse phenotyping, PBMCs were re-stimulated with 15-mer peptides covering the complete wild-type spike protein or left untreated. Sorted non-naïve CD4 + and/or CD8 + T cells were subjected to scRNAseq. Only CD4 + T cells are shown (annotation described in the methods section). The full dataset is depicted in . (B) UMAP of stimulated (blue) and unstimulated (orange) T cells (left) and Leiden clusters (right; cluster names in UMAP, cluster numbers on the right) ( n = 101,939 cells in total). Cells located within the reactive cluster are displayed with increased point size. (C) Dot plots of log-normalized expression of representative genes per cluster. Selected genes of the reactive cluster are highlighted in gray. Numbers on the left indicate cluster numbers with reactive cluster 12 highlighted in bold. (D and E) IFNG expression (D) and proliferation score (E) in unstimulated (stimulated cells in gray) and stimulated (unstimulated cells in gray) CD4 + T cells (left), and quantification in the stimulated condition of cells in the reactive cluster versus all other clusters (right). Cells located within the reactive cluster are displayed with increased point size. For IFNG , cells with log-normalized gene expression of 0 are shown in gray in UMAPs. Statistical testing by the Mann-Whitney U test. (F) UMAP visualization of cells classified as reactive (cells located in the reactive cluster or belonging to clones where at least one cell is in the reactive cluster) from donor A5 at individual time points after primary (P), secondary (S), and tertiary (T) vaccination in the stimulated condition. Color gradient indicates IFNG expression at the indicated time points. Non-reactive cells and cells from other donors are shown in gray. (G) Fraction of cells from donor A5 at each time point belonging to the reactive cluster. (H and I) Identification of spike-reactive T cells after 20h of in vitro re-stimulation of PBMCs with 15-mer peptides covering the complete wild-type spike protein. Peptides were provided in two subpools, S1 (depicted in H) and S2. Primary data (H) of donor A5 is shown. Quantification (I) of spot-forming units (SFU) for IFNγ ELISpot (combined frequencies of S1 and S2 subpools), data points represent individual donors ( n = 12–19 per time point), solid lines indicate the mean. Samples without SFU above the negative control were set to not detected (n.d.). Donor A5 is highlighted in pink. Statistical testing by the Kruskal-Wallis test followed by Dunn’s multiple comparisons test. Significant differences from the P10 time-point are indicated. ∗∗ p < 0.01, ∗∗∗∗ p < 0.0001, n.s. not significant.

Article Snippet: Plates were washed with PBS containing 0.05% Tween 20 (Sigma-Aldrich, P9416-50 mL) and incubated with biotinylated anti-human IFNγ monoclonal antibody (clone 7-B6-1, Mabtech, 3420-6-250) at 0.2 μg/well for 2 h. Plates were washed a second time with PBS containing 0.05% Tween 20 and subsequently incubated with an avidin-biotinylated peroxidase complex (VECTASTAIN Elite ABC-HRP Kit, Vector Laboratories, VEC-PK-6100) for 1–2 h. Afterward, plates were washed first with PBS containing 0.05% Tween 20 following one washing step with PBS.

Techniques: Enzyme-linked Immunospot, Functional Assay, Expressing, Gene Expression, MANN-WHITNEY, Clone Assay, In Vitro, Negative Control