Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative Airyscan-processed confocal images of HeLa STING and HeLa STING cells with stable overexpression of mEGFP-LC3B (green) treated with 120 µg/mL of cGAMP for 8 h prior to PFA-fixation and immunostaining with antibodies raised against mono- and poly-ubiquitin chains (Ub; magenta) and STING (cyan). Scale bar = 20 and 2 µm (inset). Imaging was replicated in two independent experiments. ( B ) Representative immunoblots of indicated proteins detected lysates from HeLa STING WT and HeLa STING with stable overexpression of mEGFP-LC3B prepared after treatment with 120 µg/mL cGAMP for 8 h. Immunoblotting was replicated in 3 independent experiments. ( C ) Representative immunoblots of indicated proteins detected in HeLa STING cell lysates prepared after treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( D , E ) Representative spinning disk confocal images of FRT/TREX HeLa cells stably expressing FRT/TO-DD-Vx3-EGFP, BFP-P2A-STING, and mScarletI-LC3B at the 6-hour timepoint following treatment ( D ) and quantification of the percentage (%) of cells positive for Vx3-EGFP foci over time ( E ). Cells were incubated with 1 µg/mL Doxycycline and 500 nM Shield1 for 24 h prior to treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI, and imaging every 30 min for 12 h on a spinning disk confocal microscope. Scale bar = 50 µm. Quantification is from three wells analyzed in the same experiment. Imaging was replicated in two independent experiments.

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Over Expression, Immunostaining, Ubiquitin Proteomics, Imaging, Western Blot, Stable Transfection, Expressing, Incubation, Microscopy

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: ( A ) Representative immunoblots of indicated proteins detected in THP1 cell lysates from WT and ATG16L1KO cells prepared following treatment with 1 µM diABZI for 1, 2, and 4 h. Immunoblotting was replicated in three independent experiments. ( B ) Representative immunoblots of indicated proteins detected in lysates from WT THP1 cells prepared following treatment with either DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Immunoblotting was replicated in three independent experiments. ( C , D ) Relative expression changes of indicated NFκB-related genes ( C ) and interferon-related genes ( D ) detected by quantitative RT-PCR in THP1 cells treated with DMSO, 10 µM C53, 1 µM diABZI, or both C53 and diABZI for 4 h. Quantification of relative expression is from four independent experiments analyzed at the same time. A one-way ANOVA with Tukey’s multiple comparisons test was performed on 2 −ΔΔCt values. Mean ± s.d. n = 4 *<0.05, **<0.01, ****<0.0001 ( TNF p = 0.0123; TNFAIP3 p = 0.005; IL6 p = 0.0027; IFNB1 p = <0.0001; ISG15 p = 0.0351).

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Western Blot, Expressing, Quantitative RT-PCR

Journal: The EMBO Journal

Article Title: STING induces HOIP-mediated synthesis of M1 ubiquitin chains to stimulate NF-κB signaling

doi: 10.1038/s44318-024-00291-2

Figure Lengend Snippet: Reagents and tools table

Article Snippet: STING agonist C53 , Tocris , 7741.

Techniques: Recombinant, Ubiquitin Proteomics, Sequencing, CRISPR, shRNA, Reverse Transcription, SYBR Green Assay, Control, Magnetic Beads, Negative Control, Cloning, Modification, Software