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: Developmental Cell

Article Title: Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

doi: 10.1016/j.devcel.2019.06.016

Figure Lengend Snippet: Inducers of Mitophagy in Mammalian Cells and IVM Action (A–C) HEK293 cells treated for 2 h with 15 μM IVM, for 8 h with 10 μM oligomycin and 10 μM antimycin A (OA), or for 8 h with 4 μM CCCP and stained for LC3 (A) or immunoblotted for LC3. (B) Cells treated as above, stained for TOMM20 (MITO) and WIPI2. (D) Live-cell imaging of HEK293 cells expressing CFP-LC3 and mCherry-MITO and treated with 15 μM IVM. Shown are selected time points; arrows mark mitochondrial fragments targeted by LC3. See for the whole sequence. Scale bar, 10 μm. (E–G) OCR of HEK293 cells treated with IVM. Time course and percent inhibition are plotted as shown. (H) HEK293 cells treated with 15 μM IVM and 40 μm mdiv-1 as indicated for 45 min, stained for ubiquitin, and puncta per cell determined. Means of two experiments done in duplicate are shown. (I and J) HEK293 cells treated with siRNA against DNM1L or with a non-targeting (NT) control for 72 h. After incubation with 15 μM IVM, cells were stained for ubiquitin and puncta per cell were determined. Means of two experiments done in duplicate are shown. (K) HEK-293 cells untreated or treated with 15 μM IVM for 45 min, lysed, and immunoprecipitated with ubiquitin antibodies. Samples were analyzed by mass spectrometry and the top 11 hits enriched after IVM treatment are shown. (L) Samples as in (K) were blotted for CIAP1, TRAF2, or β-COP (a loading control). (M) HEK293 cells treated with siRNA against TRAF2 or NT control for 72 h were treated as in (K) and immunoblotted for TRAF2. (N) HEK-293 cells treated with siRNA against CIAP1, CIAP2 and TRAF2 or with NT control for 96 h. After treatment with 15 μM IVM and staining for ubiquitin, puncta per cell were determined. Means of three experiments done in duplicate are shown. (O) Parallel samples were lysed and blotted for CIAP1, TRAF2 or β-COP. (P) Cells downregulated for CIAP1, CIAP2, and TRAF2 as in (N) and (O) above were incubated with IVM and the levels of mitochondrial proteins TOMM20 and MITOFUSIN 2 were determined by immunoblots and quantitated.

Article Snippet: BX-795, oligomycin, and CCCP were purchased from Tocris Biosciences.

Techniques: Staining, Live Cell Imaging, Expressing, Sequencing, Inhibition, Ubiquitin Proteomics, Control, Incubation, Immunoprecipitation, Mass Spectrometry, Western Blot

Journal: Developmental Cell

Article Title: Selective Autophagy of Mitochondria on a Ubiquitin-Endoplasmic-Reticulum Platform

doi: 10.1016/j.devcel.2019.06.016

Figure Lengend Snippet:

Article Snippet: BX-795, oligomycin, and CCCP were purchased from Tocris Biosciences.

Techniques: Recombinant, Software

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: Nucleic Acids Research

Article Title: IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway

doi: 10.1093/nar/gkaa271

Figure Lengend Snippet: IKBKE is required for AR-target gene expression and cell growth in treatment-resistant PC cells. ( A ) LNCaP-EnzR cells were reverse transfected in full media with either N/S or two independent siRNAs against IKBKE and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 3). ( B ) LNCaP-EnzR cells were treated as in (A) after 96 h, AR, TMPRSS2, FKBP5 and IKBKE expression was determined by immunoblotting and ( C ) AR, TMPRSS2, FKBP5 and IKBKE mRNA expression determined by qPCR ( n = 3). ( D ) LNCaP-EnzR cells were transfected in full media with either N/S or three pooled siRNAs against IKBKE. After 72 h, cells were re-seeded at a density of 2000 cells per well and cultured for 2 weeks. Colony forming efficiency was assessed by fixing and staining colonies with crystal violet ( n = 3). ( E ) LNCaP-EnzR cells were seeded and left to adhere and grow for 24 h, prior to treatment with either vehicle (DMSO) or the IKBKE antagonists, CAY10576 (5 μM), BX795 (5 μM), MRT67307 (5 μM), or CYT387 (5 μM) and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 2). ( F ) CWR22Rv1 cells were seeded and left to adhere and grow for 24 h, prior to treatment with either vehicle or CAY10576 (5 μM), BX795 (5 μM), MRT67307 (5 μM), or CYT387 (5 μM) and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 96 h ( n = 2). ( G ) CWR22Rv1 cells were reverse transfected with either N/S or three pooled siRNAs against IKBKE, in full media supplemented with 10 μM Enz and cell confluency assessed using the IncuCyte Zoom Live Cell Imager every 6 h for 120 h ( n = 3). 2way ANOVA, Dunnett's multiple comparisons test used in A, E, F and G **** P < 0.0001. One-way ANOVA paired Dunnetts multiple comparison test used in C and D * P < 0.05, ** P < 0.001.

Article Snippet: CAY10576 (Santa Cruz Biotechnology), MRT67307 (Stratech Scientific), BX795 (Cambridge BioScience), CYT387 (Adooq Biosciences and Cambridge BioScience), Ruxolitinib (Cambridge BioScience) and R1881 (Sigma) were stored at −80°C for no more than 6 months.

Techniques: Targeted Gene Expression, Transfection, Expressing, Western Blot, Cell Culture, Staining, Comparison