Journal: bioRxiv

Article Title: CASM potentiates STING-driven NFκB signaling in immune cells

doi: 10.64898/2026.01.21.700774

Figure Lengend Snippet: STING activation mainly drives autophagosome-independent LC3-lipidation that requires ATG16L1 lysine 490. LC3 lipidation (LC3-II FITC MFI) analyzed by flow cytometry ( A and B ) in splenocytes from WT or STING V154M/WT mutated mice ( A ) or tumor infiltrating CD4 + T cells from Fip200 fl/fl :cd4 Cre/+ mice, treated or not with i.t. injection of DMX (250 µg) for 5 h ( B ), n= 5-8 mice from 3 independent experiments, P values (*p<0.05, **p<0.01) determined by unpaired t-test. LC3 lipidation analyzed by western blot ( C-F ) in Fip200 + or ΔFip200 CD4 + T cells ( C ) stimulated or not ex vivo with DMXAA or 2’3’-cGAMP (DMX or cG) for 3 or 20 h, representative of n=2-3 independent experiments; in WT or Atg13 KO RAW264.7 macrophages ( D ) stimulated or not with DMX or cG for 3 or 20 h, representative of n=2-3 independent experiments; in adherent/myeloid cells isolated from human PBMCs ( E ) stimulated or not with cG for 3 h with or without PI3KC3/VPS34 inhibitor (PI3KC3-IN1) or Bafilomycin A1, representative of n=3 donors; in WT or ATG16L1 K490A DC2.4 cells ( F ), stimulated or not with cG for 3, 6 or 20 h, representative of n=3 independent experiments. LC3 lipidation analyzed by immunofluorescence in WT or ATG16L1 K490A DC2.4 cells ( G ) stimulated or not with cG for 3 h, quantification of LC3 dots from ten fields of view, each containing >200 cells, **** P < 0.0001, and representative images, scale bar, 2 µm. LC3 lipidation analyzed by western blot ( H and I ) in WT or ATG16L1 K490A BMDCs ( H ) or CD4 + T cells ( I ), stimulated or not with cG for 3, 6 or 20 h, representative of 2 to 6 independent experiments. LC3-II:LC3-I ratios are indicated below western blot images and increases in LC3-II:LC3-I ratios, compared to control conditions, are highlighted in bold.

Article Snippet: TBK1/IKKε inhibition was performed by pre-treating cells overnight with 0.5 μM of BX795 (TBK1/IKKε inhibitor - InvitroFitTM Invivogen, tlr-bx7) before STING agonist addition for 3 h. To distinguish between autophagy and CASM, cells were pre-treated with Bafilomycin A1 (BafA1, 100 nM, Tocris, 1334) or PIK3C3-IN1 (1 μM, MedChemExpress, HY-12795) for 1 h and co-treated with cG for 3 additional hours.

Techniques: Activation Assay, Flow Cytometry, Injection, Western Blot, Ex Vivo, Isolation, Immunofluorescence, Control

Journal: bioRxiv

Article Title: CASM potentiates STING-driven NFκB signaling in immune cells

doi: 10.64898/2026.01.21.700774

Figure Lengend Snippet: STING-driven CASM is induced by various STING ligands and requires STING but not IRF3 or RUBICON. LC3 lipidation analyzed by western blot ( A to D) in WT or Sting KO BMDCs ( A ) or CD4 + T cells ( B ), stimulated or not with DMX, cG, cdi-AMP or cdi-GMP for 3 h, representative of n=2 to 5 independent experiments; in WT or Irf3 KO BMDCs ( C ) or CD4 + T cells ( D ), stimulated or not with cG for 3 h, representative of n=3 independent experiments. LC3 lipidation (LC3-FITC MFI, normalized to Bafilomycin A1 condition) analyzed by flow cytometry ( E ) in WT or Rubcn KO CD11b + cells, CD4 + or CD8 + T cells, stimulated or not with DMX for 3 h, n= 5 mice from 3 independent experiments, P values (*p<0.05, **p<0.01) determined by two-way ANOVA. LC3-II:LC3-I ratios are indicated below western blot images and increases in LC3-II:LC3-I ratios, compared to control conditions, are highlighted in bold.

Article Snippet: TBK1/IKKε inhibition was performed by pre-treating cells overnight with 0.5 μM of BX795 (TBK1/IKKε inhibitor - InvitroFitTM Invivogen, tlr-bx7) before STING agonist addition for 3 h. To distinguish between autophagy and CASM, cells were pre-treated with Bafilomycin A1 (BafA1, 100 nM, Tocris, 1334) or PIK3C3-IN1 (1 μM, MedChemExpress, HY-12795) for 1 h and co-treated with cG for 3 additional hours.

Techniques: Western Blot, Flow Cytometry, Control

Journal: bioRxiv

Article Title: CASM potentiates STING-driven NFκB signaling in immune cells

doi: 10.64898/2026.01.21.700774

Figure Lengend Snippet: STING-associated immune responses are upregulated in autophagy-incompetent macrophages and T cells. ( A ) Heatmap representing indicated cytokine and ISG mRNA expression (Z-score) determined by RTqPCR from WT and Atg13 KO RAW264.7 macrophages stimulated or not with cG for 3 h. ( B ) IFN-β and CXCL10 secretion measured by ELISA from WT and Atg13 KO RAW264.7 macrophages stimulated or not with cG for 3 h or 6 h respectively. ( C ) Heatmap representing indicated cytokine and ISG mRNA expression (Z-score) determined by RTqPCR from Fip200 + and ΔFip200 CD4 + T cells stimulated or not with cG for 20 h. ( A and C ) Pooled data from 4 independent experiments, P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not represented) determined by two-way ANOVA from each target relative expression. ( D ) IFN-β, CXCL10 and IL-6 secretion measured by ELISA from Fip200 + and ΔFip200 CD4 + T cells stimulated or not with cG 20 h. ( B and D ), pooled data (Mean +/- SD) from 2 to 5 independent experiments, P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not represented) determined by two-way ANOVA. ( E and F ) Western blot analyses of indicated proteins and corresponding relevant quantifications from WT and Atg13 KO RAW264.7 macrophages ( E ) or Fip200 + and ΔFip200 CD4 + T cells ( F ) stimulated or not with cG for indicated time, pooled data (Mean +/- SEM) from 3 to 5 independent experiments, P values (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns not represented) determined by two-way ANOVA.

Article Snippet: TBK1/IKKε inhibition was performed by pre-treating cells overnight with 0.5 μM of BX795 (TBK1/IKKε inhibitor - InvitroFitTM Invivogen, tlr-bx7) before STING agonist addition for 3 h. To distinguish between autophagy and CASM, cells were pre-treated with Bafilomycin A1 (BafA1, 100 nM, Tocris, 1334) or PIK3C3-IN1 (1 μM, MedChemExpress, HY-12795) for 1 h and co-treated with cG for 3 additional hours.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: CASM potentiates STING-driven NFκB signaling in immune cells

doi: 10.64898/2026.01.21.700774

Figure Lengend Snippet: STING-driven CASM regulates RELA/p65-induced inflammatory response in myeloid cells. ( A ) GSEA (Gene set enrichment analysis; Normalized Enrichment Score (NES) & -log10 (p adj )) obtained from RNA sequencing analysis of WT or ATG16L1 K490A BMDCs stimulated or not with cG for 3 h. ( B ) Heatmap representing indicated cytokine and ISG mRNA expression (Z-score) determined by RTqPCR from WT or ATG16L1 K490A BMDCs stimulated or not with cG for 3 or 20 h, pooled data (Mean) from n=6-7 mice from 3 independent experiments, P values (*p<0.05) determined by two-way ANOVA from each target relative expression. IFN-β secretion measured by ELISA ( C ) or western blot analysis of indicated proteins and corresponding quantifications ( D ) from WT or ATG16L1 K490A BMDCs stimulated or not with cG for 3 ( D ) or 20 h ( C and D ), pooled data (Mean +/- SD) from n=5 mice from 2 independent experiments, P values (**p<0.01, ***p<0.001, ****p<0.0001) determined by two-way ANOVA. Tnfa mRNA expression determined by RTqPCR (Fold Change FC; Normalized to mean of each control condition) ( E ) and TNF-α secretion measured by ELISA ( F ) from WT or ATG16L1 K490A DC2.4 cells stimulated or not with cG for 6 h, pooled data (Mean +/- SD) from 3 to 5 independent experiments, P values (*p<0.05, ***p<0.001, ****p<0.0001) determined by two-way ANOVA. ( G ) Heatmap representing indicated molecule secretion (Z-score) measured by Cytokine Array from WT or ATG16L1 K490A DC2.4 cells stimulated or not with cG for 6 h, pooled data from 2 independent experiments. Western blot analysis of indicated proteins from total ( H and I ) or nuclear ( J ) extracts and corresponding quantifications (Fold Change FC, normalized to mean of WT mouse control condition) from WT or ATG16L1 K490A DC2.4 cells ( H and J ) or BMDCs ( I ) stimulated or not with cG for 3 h, pooled data (Mean & SD) from 5 independent experiments ( H ) or from n=3 mice ( I and J ), P values (*p<0.05, **p<0.01) determined by two-way ANOVA.

Article Snippet: TBK1/IKKε inhibition was performed by pre-treating cells overnight with 0.5 μM of BX795 (TBK1/IKKε inhibitor - InvitroFitTM Invivogen, tlr-bx7) before STING agonist addition for 3 h. To distinguish between autophagy and CASM, cells were pre-treated with Bafilomycin A1 (BafA1, 100 nM, Tocris, 1334) or PIK3C3-IN1 (1 μM, MedChemExpress, HY-12795) for 1 h and co-treated with cG for 3 additional hours.

Techniques: RNA Sequencing, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Biomaterials Research

Article Title: Hypochlorous Acid-Responsive Prodrug Nanoplatform for Synergistic Cancer Immunotherapy

doi: 10.34133/bmr.0300

Figure Lengend Snippet: Schematic illustration of the design and mechanism of MD1a NP for synergistic cancer immunotherapy. (A) The HOCl-responsive methylene blue (MB)–doxorubicin (DOX) dimer prodrug co-assembles with the stimulator of interferon genes (STING) agonist 1a to form a tumor-activatable nanoplatform (MD1a NP). HOCl stimulation activates and triggers the release of MB and DOX, which in turn promotes nanoparticle disassembly and facilitates the subsequent liberation of 1a. (B) Following intravenous administration, elevated intratumoral HOCl triggers the activation and the subsequent release of MB and DOX, enabling synergistic photochemotherapy under near-infrared (NIR) laser irradiation. This process induces robust immunogenic cell death (ICD) while minimizing systemic off-target effects. Concurrently, HOCl-triggered nanoparticle disassembly accelerates 1a release, activating the STING pathway and establishing an immune-promoting tumor microenvironment (TME). In orthotopic 4T1 breast cancer mouse models, MD1a NP-mediated in situ tumor vaccination (ISTV) elicited strong antitumor immunity, effectively inhibiting both primary and distant tumor growth, preventing lung metastasis, and prolonging overall survival. PDT, photodynamic therapy; DAMPs, damage-associated molecular patterns; DC, dendritic cell.

Article Snippet: All solvents were obtained from Sinopharm Chemical Reagent Co., Ltd. STING agonist 1a (HY-131994) and Cell Counting Kit-8 (CCK-8) were purchased from MedChemExpress, China.

Techniques: Activation Assay, Irradiation, In Situ