Journal: iScience

Article Title: Cetylpyridinium chloride triggers paraptosis to suppress pancreatic tumor growth via the ERN1-MAP3K5-p38 pathway

doi: 10.1016/j.isci.2024.110598

Figure Lengend Snippet:

Article Snippet: Rabbit recombinant polyclonal anti-ATG7 , Proteintech Biotechnology , Cat# 10088-2-AP; RRID: AB_2062351.

Techniques: Recombinant, Lysis, Modification, Polyacrylamide Gel Electrophoresis, Protease Inhibitor, CCK-8 Assay, Sequencing, Software

Journal: Cells

Article Title: Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells

doi: 10.3390/cells13060531

Figure Lengend Snippet: ATG7-independent autophagy flux in gingival epithelial cells (GEC). ( A ) Western blot analysis of ATG7 knockout in WT and T2R14KO GECs. ATG7 KD in WT and T2R14 KO GECs was performed using an shRNA lentiviral approach. WT denoted WT-sc shRNA, and T2R14KO denoted T2R14 KO sc shRNA. ( B – D ) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Flow cytometry evaluation of the red, green, and red/green fluorescence ratio for detecting the autophagy flux in ATG7KD and T2R14KO-ATG7KD in GECs under serum-starved conditions and their respective normal growth condition. The bar graphs were generated using GraphPad PRISM 9.0. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post hoc test * p < 0.05, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant. The data represent the SEM of 3–4 independent experiments. ( E ) Acridine Orange staining of AVOs for detecting autophagy flux in GECs. Representative raw traces showing the flow cytometric detection of red and green fluorescence in Acridine Orange-stained GECs (ATG7KD and T2R14-ATG7KD) that were serum-starved for 72 h for detecting the autophagy flux.

Article Snippet: The following antibodies were purchased: mouse monoclonal anti-β-actin (#A5441) from Sigma Aldrich (Oakville, ON, Canada), goat anti-rabbit IgG-HRP conjugate (#17-6515) from Bio-Rad (Mississauga, ON, Canada), goat anti-mouse IgG-HRP conjugate (#A-10668), and rabbit polyclonal anti-T2R14 (#OSR00161W) from Thermo Fischer Scientific, Carlsbad, CA, USA, and rabbit polyclonal ATG7 from cell signaling.

Techniques: Western Blot, Knock-Out, shRNA, Staining, Flow Cytometry, Fluorescence, Generated

Journal: Cells

Article Title: Bitter Taste Receptor T2R14 and Autophagy Flux in Gingival Epithelial Cells

doi: 10.3390/cells13060531

Figure Lengend Snippet: Intracellular calcium mobilization assay in GECs. ( A ) Pharmacological characterization of autophagy flux inhibitor, Baf A1 (10 nM) and autophagy inducer Rapa (1000 nM) on the calcium mobilization in GECs. WT, T2R14KO, ATG7 KD, and T2R14KO-ATG7KD cells (approx. 30,000/96 wells) were loaded with Fluo-4NW calcium dye and the intracellular calcium response was measured after Baf A1 and Rapa treatment. The data shown are the mean ± SEM of three independent experiments performed in triplicate. ( B ) Effect of S. aureus autoinducer peptide (AIP-1) and S. mutans competence-stimulating peptide (CSP-1) on calcium mobilization in GECs. Intracellular calcium release measurement after the application of autoinducer peptides (AIP from S. aureus and CSP-1 from S. mutans (50 µM each) in GECs. Results are from three independent experiments performed in triplicate. ( C ) Combined effect of Baf A1 and CSP-1 on calcium signaling in GECs. WT, T2R14KO, ATG7 KD, and T2R14KO-ATG7KD cells (approx. 30,000/96 wells) were loaded with Fluo-4NW calcium dye and the intracellular calcium response was measured after CSP-1 and CSP-1 plus Baf A1 treatment. The data shown are the mean ± SEM of three independent experiments performed in triplicate. Statistical significance was calculated using a one-way ANOVA with Bonferroni’s post -hoc test * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 and ns denotes non-significant.

Article Snippet: The following antibodies were purchased: mouse monoclonal anti-β-actin (#A5441) from Sigma Aldrich (Oakville, ON, Canada), goat anti-rabbit IgG-HRP conjugate (#17-6515) from Bio-Rad (Mississauga, ON, Canada), goat anti-mouse IgG-HRP conjugate (#A-10668), and rabbit polyclonal anti-T2R14 (#OSR00161W) from Thermo Fischer Scientific, Carlsbad, CA, USA, and rabbit polyclonal ATG7 from cell signaling.

Techniques: Calcium Mobilization Assay