Journal: bioRxiv

Article Title: STING activation depends on ACBD3 and other phosphatidylinositol 4-phosphate-regulating proteins

doi: 10.1101/2022.10.17.512580

Figure Lengend Snippet: PI4KB and OSBP inhibition have opposite effects on STING activation a. THP-1 cells were pre-incubated with the PI4KB inhibitor BF738735 (10uM) and subsequently stimulated with the STING agonist 2’3’-RR CDA (1.67ug/ml). After 20-24h, tdTomato-reporter expression was quantified by flow cytometry. b. (left panel) PI4KB mRNA expression levels in THP-1 cells expressing a control gRNA or gRNAs targeting PI4KB. Mean ± SEM of n = 3 technical replicates are shown. (right panel) Cells were stimulated with 2’3’-RR CDA for 20-24h and tdTomato reporter expression was quantified by flow cytometry. Mean ± SEM of n = 3 biological replicates are shown. c. 293T cells expressing eGFP-mSTING were transfected with a phosphatase-dead Sac1 (inactive), active Sac1 wt, or Sac1-kkaa mutant and stimulated or not with 2’3’-RR CDA (RR-CDA) for 8h. After stimulation, cells were stained for phospho-STING and analyzed by flow cytometry. Mean ± SEM of n = 3 biological replicates are shown. d. THP-1 cells were preincubated with DMSO or the OSBP inhibitors itraconazole (ITZ) or OSW-1 and stimulated with 2’3’-cGAMP (7μg/ml) or left untreated. Reporter expression was quantified by flow cytometry 20h after stimulation. Representative images of n = 3 biological replicates are shown. e. Mean fluorescence intensity of tdTomato reporter in THP-1 cells stimulated with STING agonists for 20h-24h in the presence of DMSO, itraconazole (ITZ), or OSW-1. f. IFNB1 or CXCL10 mRNA levels in THP-1 cells pre-treated with DMSO or itraconazole (ITZ) for 1h and stimulated with 2’3’-RR CDA (RR-CDA) for 4 or 24h. g. CXCL10 mRNA levels in THP-1 cells expressing a control gRNA or STING gRNA (STING knockdown; KD) treated as in e and measured after 24h. h. CXCL10 mRNA levels in THP-1 pre-treated as in e and transfected with VACV-70 dsDNA for 4h or 24 e-h. Mean ± SEM of n = 3 biological replicates are shown.

Article Snippet: Reagents used include itraconazole (Santa Cruz Biotechnology cat. no. sc-205724A), OSW-1 (a kind gift from M. Shair, Harvard University), BF738735 (Tocris cat.no.

Techniques: Inhibition, Activation Assay, Incubation, Expressing, Flow Cytometry, Control, Transfection, Mutagenesis, Staining, Fluorescence, Knockdown

Journal: bioRxiv

Article Title: STING activation depends on ACBD3 and other phosphatidylinositol 4-phosphate-regulating proteins

doi: 10.1101/2022.10.17.512580

Figure Lengend Snippet: OSBP inhibition increases STING activation and decreases STING degradation a. Immunoblot analysis of the indicated (phosphorylated) proteins expressed by THP-1 cells. Cells were pre-treated with DMSO (D) or itraconazole (I) for 1h and stimulated with 2’3’-cGAMP (20ug/ml) for the indicated time points. Representative images of n = 3 biological experiments are shown. b. Immunofluorescence live-cell imaging eGFP-tagged mouse-STING in 293T cells pre-treated for 1h with DMSO or itraconazole (ITZ) and stimulated with 2’3’-cGAMP for 15h. Representative images of n = 2 biological experiments are shown. c. The number of STING clusters/GFP+ cells shown in c was quantified over time using the ‘particle analysis’ function of ImageJ. Representative quantification of n = 2 biological experiments is shown. d. Expression of eGFP-tagged mouse STING in THP-1 cells pre-treated with DMSO or itraconazole for 1h and stimulated with 2’3’-RR CDA (10μg/ml) or left unstimulated for 20h. Representative image of n = 3 biological replicates is shown. e. Quantification of eGFP-STING in THP-1 cells shown in e. Mean ± SEM of n = 3 biological replicates are shown. f. tdTomato reporter expression of THP-1 cells pre-treated with the PI4KB inhibitor (PI4KBi) BF738735 for 1h followed by pre-treatment with itraconazole (ITZ) for 1h and stimulation with 2’3’-cGAMP for 20h. Mean ± SEM of n = 2 biological replicates are shown.

Article Snippet: Reagents used include itraconazole (Santa Cruz Biotechnology cat. no. sc-205724A), OSW-1 (a kind gift from M. Shair, Harvard University), BF738735 (Tocris cat.no.

Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Live Cell Imaging, Particle Size Analysis, Expressing

Journal: JOM (Warrendale, Pa. : 1989)

Article Title: Use of Drawing Lithography-Fabricated Polyglycolic Acid Microneedles for Transdermal Delivery of Itraconazole to a Human Basal Cell Carcinoma Model Regenerated on Mice

doi: 10.1007/s11837-016-1841-1

Figure Lengend Snippet: Microneedle-facilitated delivery of itraconzole to regenerated human basal cell carcinoma. Skin grafts expressing Sonic Hedgehog were generated on immunodeficient mice; topical treatment of itraconazole was facilitated by either four 1 × 4 polyglycolic acid microneedle arrays or stainless steel microneedle arrays. Hematoxylin and eosin staining of 4-week-old regenerated human skin grafts of: (a) untreated control, (b) polyglycolic acid microneedle array-facilitated delivery of itraconazole-containing solution, and (c) stainless steel microneedle array-facilitated delivery of itraconazole-containing solution.

Article Snippet: Preparation of the Itraconazole-Containing Solution Itraconazole 99% (Acros Organics, NJ, USA), dimethylsulfoxide 99% (FWI, Tulsa, OK, USA), polyethylene Glycol 400 (Letco, Decatur, AL, USA), and a dimethylsulfoxide safe 0.22- μ m sterilizing filter (Pall, Port Washington, NY, USA) were used to prepare the itraconazole-containing solution.

Techniques: Expressing, Generated, Staining, Control

Journal: Clinical chemistry and laboratory medicine

Article Title: Simultaneous quantitation of five triazole anti-fungal agents by paper spray-mass spectrometry.

doi: 10.1515/cclm-2019-0895

Figure Lengend Snippet: Figure 1: Overlays of calibration curves for the five triazoles: fluconazole (A), voriconazole (B), posaconazole (C), itraconazole (D), and hydroxyitraconazole (E). Data were collected over 7 different days (one calibration curve/day). Linearity ranged from R2 = 0.94–0.99.

Article Snippet: Hydroxyitraconazole and hydroxyitraconazole-D5 were purchased from Fitzgerald Industries International (Acton, MA, USA) and Toronto Research Chemicals Incorporated (Ontario, Canada).

Techniques:

Journal: Food chemistry

Article Title: Glucosinolate profile and specifier protein activity determine the glucosinolate hydrolysis product formation in kohlrabi (Brassica oleracea var. gongylodes) in a tissue-specific way.

doi: 10.1016/j.foodchem.2024.142032

Figure Lengend Snippet: Fig. 6. Hierachial clustering and heatmap visualization of selected data from three biological replicates for each of the nine kohlrabi tissues analyzed in the first replicate experiment. Represented is the correlation between metabolites (glucosinolates and glucosinolate hydrolysis products), myrosinase activity, proportion of nitriles and isothiocyanates formed (% CN and % ITC), ESP activity (% CETP), total nitrile-specifier protein abundance (sum NSP), total epithiospecifier protein abundance (sum ESP), as well as BoESM1 and BoESM1-like abundance in each biological replicate. The heatmap was generated by Metaboanalyst 6.0 (Pang et al., 2024). The data was normalized, the features autoscaled and the Euclidean distance measure and Ward clustering algorithm applied.

Article Snippet: 3-(Methylsulfinyl)propyl ITC (3MSOP-ITC) and 4-(methylthio) butyl ITC (4MTB-ITC, ≥ 98 %) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activity Assay, Quantitative Proteomics, Generated