Journal: Cell Chemical Biology

Article Title: Identifying enhancers of innate immune signaling as broad-spectrum antivirals active against emerging viruses

doi: 10.1016/j.chembiol.2022.05.009

Figure Lengend Snippet:

Article Snippet: BX-795 , MedChemExpress , Cat# HY-10514 CAS: 702675-74-9.

Techniques: Recombinant, Cell Viability Assay, Luciferase, Derivative Assay, Software, Transfection

Journal: Psychiatry research

Article Title: Genetic variations in evolutionary accelerated regions disrupt cognition in schizophrenia.

doi: 10.1016/j.psychres.2022.114586

Figure Lengend Snippet: Figure 4. BAFFR regulates the expression of PTEN and FOXO1 (A) Western blot analysis of PTEN and FOXO1 in naive and MBCs treated for 24–72 h ± BAFF (20 ng/mL). N-fold changes of PTEN and FOXO1 expression were calculated as signals normalized to actin and further normalized to untreated samples from day 0 per subset. The experiment was performed once in this format. (B) Flow cytometric analysis of PTEN expression in naive and MBCs treated for 24–72 h ± BAFF. N-fold change of PTEN MFI was calculated as [(PTEN MFI + BAFF)/(PTEN MFI –BAFF)]. (C) Western blot analysis of phosphorylated FOXO1 (S256) in naive B cells treated with BAFF or F(ab0)2 anti-IgM (2 mg/mL) for 0.5–8 h. N-fold change in pFOXO1 levels was calculated as described in (A). (D) Western blot analysis of PTEN in naive B cells treated with inhibitors against SYK (2.5 mM R406), PI3Kd (0.625 mM nemiralisib), PDK1 (5 mM BX-795), NIK (10 mM SMI1), or DMSO (control) ± BAFF for 2 days. (E) Western blot analysis of FOXO1 in naive B cells treated with iSYK, iNIK, or DMSO ± BAFF for 2 days. (D and E) Blots are representative of R2 independent experiments. N-fold changes were calculated as signals normalized to actin and further normalized to the control untreated sample. (F) Flow cytometric analysis of CD62L and CXCR4 in naive and MBCs treated overnight with BAFF in the presence of FOXO1 inhibitor (2.5 mM AS1842856) or DMSO (control). Plots show the mean of CD62L and CXCR4 MFI from 2 independent experiments (2–3 replicates/experiment, 2 HDs). *p < 0.05; **p < 0.01; ****p < 0.0001; ns, not significant (2-way ANOVA with Tukey multiple comparisons test).

Article Snippet: Chemicals, peptides, and recombinant proteins BAFF Smulski et al., 2017 N/A CD40L Smulski et al., 2017 N/A SARS-Cov2 spike protein Thermofisher Cat# RP-87680 SYK inhibitor (R406) Selleckchem Cat# S2194; CAS 841290-81-1 Dual BCR-ABL/LYN inhibitor (Bafetinib/INNO-406) Selleckchem Cat# S1369; CAS 859212-16-1 PI3Kd inhibitor (nemiralisib/GSK2269557) Selleckchem Cat# S7937; CAS 1254036-71-9 PDK1 inhibitor (BX-795) Selleckchem Cat# S1274; CAS 702675-74-9 FOXO1 inhibitor (AS1842856) Selleckchem Cat# S8222; CAS 836620-48-5 NIK inhibitor (SMI1) Hycultec Cat# HY112433; CAS 1660114-31-7 human monoclonal IgG1l anti-BAFF antibody belimumab (Benlysta) GlaxoSmithKline Pharmaceuticals CAS 356547-88-1 hTACI (aa 31–110)-hIgG1 Fc (aa 245–470) Kowalczyk-Quintas et al., 2019 N/A Fixable viability dye eFluor450 eBioscience Cat# 65-0863-14 Brefeldin A Biolegend Cat# 420601 Critical commercial assays Neon Transfection System 10 mL Kit Thermo Fischer Scientific Cat# MPK1096 NEBuilder HiFi DNA Assembly Cloning Kit New England Biolabs Cat# E5520S jetPEI Polyplus transfection Cat# 101-10N Experimental models: Cell lines DG-75 DSMZ Cat# ACC 83 HEK 293T DSMZ Cat# ACC 635 Oligonucleotides 5ʹ-TCCTCCATGGCAACTACACGTGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79A.1.AB 5ʹ-AACACCTCGGAGGTCTACCAGGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD79B.1.AA 5ʹ-CCTTCCAAGGACGTCATGCAGGGC-3ʹ Integrated DNA Technologies N/A 5ʹ-TTAATAACATCACCATGCACAGG-3ʹ Integrated DNA Technologies Hs.Cas9.LYN.1.AE 5ʹ-CTCACCGTCCTTGTCTCCGTCGG-3ʹ Integrated DNA Technologies N/A 5ʹ-GTTTGGAAGAGGATCACACTCGG-3ʹ Integrated DNA Technologies Hs.Cas9.CD27.1.AF 5ʹ-CAAATACACGAACTTCTCCC-3ʹ Integrated DNA Technologies Hs.Cas9.TCL1A.1.AE 5ʹ-CTCGGGAAGGTACCAAGGAT-3ʹ Integrated DNA Technologies Hs.Cas9.TNFRSF13B.1.AA Software and algorithms Flow Jo_v10 Flow Jo https://www.flowjo.com/solutions/flowjo RRID: SCR_008520 GraphPad Prism 8.4 GraphPad https://www.graphpad.com/scientific- software/prism RRID: SCR_002798 Image J V.2.3.0 Rasband, 1997-2018 http://imagej.net/ImageJ RRID: SCR_003070 ClustVis Metsalu and Vilo, 2015 https://biit.cs.ut.ee/clustvis/; RRID: SCR_017133 String Szklarczyk et al., 2020 https://www.string-db.org; RRID: SCR_005223 Cell Reports 39, 111019, June 28, 2022 e3

Techniques: Expressing, Western Blot, Control

Journal: EMBO Reports

Article Title: DSTYK phosphorylates STING at late endosomes to promote STING signaling

doi: 10.1038/s44319-025-00394-9

Figure Lengend Snippet: ( A , B ) TBK1 is essential for the activation of STING signaling. DSTYK -knockout HeLa cells, TBK1 -knockout HeLa cells, DKO HeLa cells and control cells were stimulated with pDNA transfection (1 μg/mL) for 12 h, followed by detection of phosphorylation of IRF3 and STING at Ser366 via western blotting ( A ) and IFNB production via quantitative PCR ( B ; n = 3 biological replicates). ( C , D ) TBK1 regulates the function of DSTYK to STING. DSTYK- knockout HeLa cells stably expressing DSTYK (DSTYK KO-F-DK), DKO HeLa cells stably expressing DSTYK (DKO-F-DK) and control cells were stimulated with pDNA transfection (1 μg/mL) for 12 h, followed by detection of phosphorylation of IRF3 and STING at Ser366 via western blotting ( C ) and IFNB production via quantitative PCR ( D ; n = 3 biological replicates). ( E ) Inhibition of TBK1 kinase activity restricts the function of DSTYK. DSTYK -knockout HeLa cells stably expressing DSTYK (DSTYK KO-F-DK) and other control cells were pretreated with BX795 (10 μM) for 4 h and then stimulated with cGAMP (100 nM) for 30 min, followed by detection of phosphorylation of IRF3 and STING at Ser366 via western blotting. ( F ) Quantification of the colocalization coefficients between STING and different organelles in Fig. . Quantification of the colocalization coefficients between STING and organelles was analyzed in ImageJ ( n = 20 biological replicates). ( G ) TBK1 controls colocalization between STING and DSTYK. DSTYK -knockout HeLa cells stably expressing DSTYK (DSTYK KO-F-DK) and DKO HeLa cells stably expressing DSTYK (DKO-F-DK) were stimulated with pDNA transfection (1 μg/mL) for 3, 12 and 24 h, followed by confocal imaging to observe STING and DSTYK colocalization with GRASP65 and LAMP1, respectively; scale bars, 20 μm. ( H ) TBK1 controls STING post-Golgi trafficking via its kinase activity. WT HeLa cells were treated with DMSO, 2.5 μM BX795 and 5 μM BX795 for 1 h, then stimulated with pDNA transfection (1 μg/mL) for 3 and 24 h, followed by confocal imaging to observe STING colocalization with GRASP65 and Rab7, respectively; scale bars, 20 μm. Quantification of the indicated band intensities in ( A , C , E ) was performed in ImageJ, and quantification results are labeled below the indicated bands ( n = 3 technical replicates). Data are representative of at least three independent experiments. Data in ( B , D , F ) are shown as the mean ± SD. P values were determined by two-way ANOVA and the exact P values are shown in the figures. .

Article Snippet: BX795 , TargetMol , T1830.

Techniques: Activation Assay, Knock-Out, Control, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Inhibition, Activity Assay, Imaging, Labeling

Journal: EMBO Reports

Article Title: DSTYK phosphorylates STING at late endosomes to promote STING signaling

doi: 10.1038/s44319-025-00394-9

Figure Lengend Snippet: ( A , B ) TBK1 knockout inhibits STING post-Golgi trafficking. TBK1 -knockout HeLa cells, DSTYK- knockout HeLa cells and control cells were stimulated with pDNA (1 μg/mL) for the indicated amounts of time, followed by confocal imaging to observe STING colocalization with GRAPS65 ( A ) and Rab7 ( B ); scale bars, 10 μm. ( C ) TBK1 knockout inhibits STING degradation. TBK1 -knockout HeLa cells, DSTYK- knockout HeLa cells and control cells were stimulated with pDNA (1 μg/mL) for 36 h, and STING protein level was detected via western blotting. ( D ) BX795 inhibits STING signaling. WT HeLa cells were treated with DSMO, BX795 (2.5 μM, 5 μM) for 1 h, then stimulated with pDNA for 24 h, and phosphorylation of IRF3 and STING Ser366 was detected by western blotting. Data are representative of at least three independent experiments.

Article Snippet: BX795 , TargetMol , T1830.

Techniques: Knock-Out, Control, Imaging, Western Blot

Journal: EMBO Reports

Article Title: DSTYK phosphorylates STING at late endosomes to promote STING signaling

doi: 10.1038/s44319-025-00394-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: BX795 , TargetMol , T1830.

Techniques: Recombinant, Sequencing, shRNA, Transfection, Real-time Polymerase Chain Reaction, SYBR Green Assay, Mutagenesis, Software

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K, PDK1, and Src regulation of nuclear YAP via Lats in serum-starved, subconfluent cells. (A) PI3K and PDK1 inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNAs were serum starved and treated with DMSO (solvent control), 10 µM wortmannin (PI3K inhibitor), or 5 µM BX-795 (PDK1 inhibitor) for 30 min. On-target plus nontargeting pool was used as a control siRNA. Localization of endogenous YAP was identified by immunofluorescence staining. (B) SFK inhibitors and YAP localization. Serum-starved, low cell density MCF-10A cells were incubated with SFK inhibitors (10 µM each of PP2, dasatinib, SKI-1, and SU6656) for 30 min. 10 µM each of PP3 and imatinib were used as controls. YAP subcellular localization was determined by immunofluorescence staining. Alexa Fluor 594 secondary antibody was used for SU6656, which has high background green fluorescence. (C) Biochemical effects of Src inhibition. PP3- or PP2-treated MCF-10A cells were analyzed by Western blot using anti-YAP and anti–phospho-YAP (S127) antibodies. Phosphorylated YAP was detected by mobility shift on Phos-tag SDS-PAGE. (D) SFK inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNA were serum starved and treated with 10 µM PP3 or PP2. After 30 min, cells were fixed for immunofluorescence staining with anti-YAP antibody. (E) Depletion of individual SFK. MCF-10A cells were transfected with control, Src, Fyn, or Yes siRNA. After serum starvation, subcellular localization of endogenous YAP was identified by immunofluorescence staining and quantified based on the criteria shown under the graph. More than 120 cells from four random views were quantified. (F) Src knockdown relative to Lats. MCF-10A cells were transfected with control, Src, Lats1/2, or combined siRNA of Src and Lats1/2. Cells were serum starved for 24 h before fixation and stained with anti-YAP antibody. (A, B, and D–F) One of three independent results is presented. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Transfection, Control, Solvent, Immunofluorescence, Staining, Incubation, Fluorescence, Inhibition, Western Blot, Mobility Shift, SDS Page, Knockdown

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K–PDK1, but not FAK, act downstream of Src. MCF-10A cells expressing doxycycline-inducible CA-Src-GFP fusion protein were treated with 1 µg/ml doxycycline (Dox) in complete medium for 12 h and starved for 24 h in starvation medium containing doxycycline. Indicated inhibitors were added to culture medium and incubated for 30 min before fixation. Subcellular localization of YAP was determined by immunofluorescence staining. One of three independent results is presented. Tet, tetracycline (or doxycycline) inducible. Bar, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Expressing, Incubation, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: Attachment to fibronectin-coated coverslips induces YAP nuclear accumulation via the FAK–Src–PI3K–PDK1 pathway. (A) Attachment to fibronectin, poly- d -lysine, or laminin. Serum-starved MCF-10A cells were dissociated with Accutase and sparsely seeded on fibronectin (FN)-, poly- d -lysine (PL)–, or laminin (LN)-coated coverslips in starvation medium. Cells were incubated for 2 h before fixation. Subcellular localization of YAP was identified by immunofluorescence staining and quantified based on the criteria shown in . More than 170 cells from eight random views were quantified. DAPI staining was used to locate the nucleus. We performed three independent experiments. (B) Effects of inhibitors and growth factors on attachment-induced YAP nuclear localization. Serum-starved MCF-10A cells were detached and seeded on fibronectin- or poly- d -lysine–coated coverslips in starvation medium. After 3 h of incubation, cells were treated with the indicated inhibitors or mitogens for 30 min. Subcellular localization of YAP was identified by immunofluorescence staining. More than 120 cells from eight random views were quantified and confirmed in three independent experiments. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Incubation, Immunofluorescence, Staining

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: Upstream negative regulators of the Hippo pathway. Soluble growth factors including EGF and LPA inactivate the growth-inhibitory Hippo pathway through the stimulation of PI3K–PDK1 signaling. EGFR, but not LPA, signaling depends on Src kinase. Integrin receptors bind to fibronectin (FN), and mechanical stimulus triggers FAK-Src signaling and leads to the activation of YAP in a PI3K–PDK1-dependent manner.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Activation Assay

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