Journal: Cells

Article Title: Photochemotherapy Induces Interferon Type III Expression via STING Pathway

doi: 10.3390/cells9112452

Figure Lengend Snippet: The Stimulator of Interferon Genes (STING) pathway is activated in Hut78 by 8–MOP + UVA treatment. Downregulation of alleged pathway elements by specific small interfering RNA (siRNA) or by a chemical inhibitor result in decreased IFNL1 expression. Expression of IFNL1 following 8–MOP + UVA treatment combined with ( A ) STING downregulation by siRNA, ( B ) cyclic GMP-AMP synthase (cGAS) downregulation by siRNA, ( C ) TBK1 inhibition by BX795 chemical inhibitor, ( D ) IRF3 downregulation by siRNA and ( E ) IRF1 downregulation by siRNA. Cell viability for respective treatments is presented in ( F ) for STING-siRNA, ( G ) for cGAS-siRNA, ( H ) for BX795-mediated TBK1 inhibition, ( I ) for IRF3-siRNA and ( J ) for IRF1-siRNA. ( K ) Transfection efficiencies for various siRNAs. Error bars represent ± SEM of the indicated N repeats. Statistics—normal distribution, paired t -test: ( A – E ) and skewed distribution, paired Wilcoxon: (F–J) . Choice of the statistical test was made based on the type of data distribution (see Methods). * p < 0.1, ** p < 0.05, ns–not significant.

Article Snippet: TBK1 inhibitor BX795 (Tocris, Abingdon, UK) and ataxia-telangiectasia and Rad3-related (ATR) kinase inhibitor AZD6738 (Selleckchem, Houston, TX, USA) were added immediately after UVA irradiation.

Techniques: Small Interfering RNA, Expressing, Inhibition, Transfection

Journal: Biomolecules & Therapeutics

Article Title: Identification of Small GTPases That Phosphorylate IRF3 through TBK1 Activation Using an Active Mutant Library Screen

doi: 10.4062/biomolther.2022.119

Figure Lengend Snippet: IRF3 phosphorylation mediated by small GTPases require TBK1. (A) HEK293 cells were co-transfected with 0.2 µg of FLAG-IRF3 and 0.2 µg of GTPases, respectively. 24 h post transfection, the cells were treated with BX795 for 3 h. (B) HEK293 cells were transfected with 0.2 µg of FLAG-IRF3 and 0.2 µg of HA-RHEB-Q64L or empty plasmid. 24 h post transfection, the cells were treated with Rapamycin or vehicle for 3 h. (C) Overall scheme of this study.

Article Snippet: BX795 (#14932) was from Cayman (Ann Arbor, MI, USA).

Techniques: Phospho-proteomics, Transfection, Plasmid Preparation

Journal: eLife

Article Title: MicroRNA-deficient mouse embryonic stem cells acquire a functional interferon response

doi: 10.7554/eLife.44171

Figure Lengend Snippet: ( a, b ) Susceptibility of Dgcr8 -/- , Dicer -/- and parental cells to TMEV infection after inhibition of IRF3 (BX795) ( a ) and Nf-κB (BMS345541) ( b ), normalized to mock-treated cells. ( c ) Heat map of significantly differentially expressed proteins (p<0.05) in the absence ( Dgcr8 -/- ) or presence ( Dgcr8 resc ) of miRNAs identified by STRING analysis. ( d ) Western blot analysis of MAVS expression in miRNA-deficient cells ( Dgcr8 -/- and Dicer -/- , lanes 2 and 5), wild-type counterparts ( Dgcr8 +/+ and Dicer +/+ , lanes 1 and 4) and respective rescued ESCs lines ( Dgcr8 resc and Dicer resc , lanes 3 and 6). MAVS quantification normalized to Tubulin and relative to wild-type levels is shown at the top of the panel. 10.7554/eLife.44171.010 Figure 3—source data 1. Source data of mass spectrometry results.

Article Snippet: Cells were pre-incubated with the inhibitors BX795, which blocks the phosphorylation of the kinases TBK1 and IKKε, and consequently IRF3 activation and IFN-β production (10 μM, Synkinase) and the inhibitor BMS345541, which targets IKβα, IKKα and IKKβ and consequently NF-κβ signalling (10 μM, Cayman Chemical) for 45 min before infection with TMEV.

Techniques: Infection, Inhibition, Western Blot, Expressing, Mass Spectrometry