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