Journal: Frontiers in Immunology

Article Title: Discovering single cannabidiol or synergistic antitumor effects of cannabidiol and cytokine-induced killer cells on non-small cell lung cancer cells

doi: 10.3389/fimmu.2024.1268652

Figure Lengend Snippet: Characterization of intrinsic alternation in the CIK cells in the CBD-inducing culture by flow cytometric analysis. (A) CIK cells were exposed to either CBD or CBD combined with the TRPV2 channel antagonist tranilast at 37°C for 1 minute. Dead cells were gated and excluded by Hoechst 33258 for Fluo-4 AM expression. (B) The phosphorylation levels of Erk1/2 in CIK cells were determined by flow cytometry. Dead cells were gated and excluded by Zombie Aqua™ dye for intracellular expression. The rabbit of isotype controls is indicated. The percentage of FITC anti-ERK1/2 Phospho (Thr202/Tyr204) was analyzed using the Flowjo V10 software. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. CIK combined with DMSO control. All data are shown as the mean ± SD, representative of four independent experiments. Statistical analysis was performed using a two-way ANOVA followed by Dunnett’s multiple comparisons test by GraphPad Prism software version 9.0.0. CIK cells were derived from 4 donors.

Article Snippet: In order to detect TRPV2 expression, the anti-TRPV2 polyclonal rabbit antibody (EpigenTek, Farmingdale, NY, U.S.A) was diluted 1:200, and the secondary antibody Donkey Anti-Rabbit IgG NorthernLightsTM NL557-conjugated antibody (R&D Systems, Inc., Minneapolis, Minnesota, U.S.A.) was diluted 1:500 in dilution buffer (1% BSA, 0.3% Triton in PBS).The slide was finally stained with a diluted DAPI solution.

Techniques: Expressing, Phospho-proteomics, Flow Cytometry, Software, Control, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Association of TRPV2 expression with apoptosis-related gene sets. (A) Distribution on TCGA-STAD GC samples based on TRPV2 expression. Green and red lines define the lower and upper tertiles, respectively. (B) and (C) Apoptosis-related Ontology and Canonical Pathways gene sets significantly enriched in samples characterized by high TRPV2 expression. Ratio indicates, among all DEGs, the percent of upregulated genes included in each gene set.

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Association of TRPV2 expression with resistance to cisplatin-induced cell death in GC cell lines. (A) Relative qRT-PCR quantification of TRPV2 mRNA in GC cell lines by using three reference genes. TRPV2 expression is reported as FC relative to that in the AGS cell line. In the lower panel, western blot analysis of TRPV2 expression in the three GC cell lines. (B) Assessment of apoptosis and necrosis rates in several GC cell lines after treatment with different concentrations of cisplatin. Statistical significance was estimated by ANOVA test and Tukey’s post hoc test on three replicates. (C) Schematic representation of correlation between TRPV2 expression and cisplatin resistance of different GC cell lines. 3REF: mean of fold change (FC) from the different housekeeping genes.

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Effect of TRPV2 chemical inhibition on intracellular calcium levels. (A) Representative flow cytometry dot plots of calcium green-1-AM staining to measure intracellular calcium levels. (B) Estimation of mean fluorescence intensity differences measured by flow cytometry among the tranilast-treated groups and control. t -test was used to perform multiple comparisons on three replicates. (C) Fluorescence microscopy evaluation of intracellular calcium levels in the presence of ionomycin (5 µM) and different concentrations of TRPV2 inhibitor (×20 magnification, Leica SP8 microscope).

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Inhibition, Flow Cytometry, Staining, Fluorescence, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Effect of TRPV2 chemical inhibition on apoptosis rates of KATO-III cells. (A) Representative dot plots obtained by flow cytometry measurement of Annexin V exposure and propidium iodide (PI) fluorescence in different culture conditions. (B) Statistical analysis on DMSO normalized counts (fold changes) of the different types of cell death by one-sample t -test, ANOVA, and Tukey’s post hoc tests on three replicates. (C) Representative western blot of apoptosis activation markers in KATO-III cells treated with cisplatin, tranilast, or their combination. (D) Statistical analysis on log 2 -transformed actin-normalized densitometry data of common apoptosis markers amounts relative to cells treated with vehicle. ANOVA and Tukey’s tests were used to assess differences among means of treated cells, whereas one-sample t -test was used for comparison with the vehicle. Data were obtained from triplicates.

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Inhibition, Flow Cytometry, Fluorescence, Western Blot, Activation Assay, Transformation Assay

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Exogenous expression of TRPV2 calcium channel in cisplatin-sensitive GC cell line. (A) Schematic representation of TRPV2 expression construct and evaluation of TRPV2 overexpression by ddPCR. (B) Representative images of TRPV2 exogenous expression by confocal microscopy in transformed AGS cell line pools as compared with the KATO-III cell line. (C) Quantification of TRPV2 expression in cells carrying the empty vector (p1_ and p2_eVect) or TRPV2 CDS (p1_ and p2_TRPV2) and comparison with KATO-III as reference. t -test on mean fluorescence intensity from three areas (A1, A2, and A3) was used for comparisons. Confocal specimen staining: NucBlue, rabbit anti-human TRPV2 revealed with goat anti-rabbit IgG-FITC; magnification: ×63; Leica SP8 microscope.

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Expressing, Construct, Over Expression, Confocal Microscopy, Transformation Assay, Plasmid Preparation, Fluorescence, Staining, Microscopy

Journal: Frontiers in Pharmacology

Article Title: Cationic Channel TRPV2 Overexpression Promotes Resistance to Cisplatin-Induced Apoptosis in Gastric Cancer Cells

doi: 10.3389/fphar.2021.746628

Figure Lengend Snippet: Effect of exogenous expression of TRPV2 calcium channel in the cisplatin-sensitive AGS GC cell line. Cisplatin-induced apoptosis in transformed AGS cell line pools expressing exogenous TRPV2 was assessed by flow cytometry. t -test was used to assess the significance of comparison between mean fold changes from three replicates (solid and dashed lines distinguish between the first and second pool).

Article Snippet: Cells were then incubated overnight at 4°C with rabbit anti-human TRPV2 primary antibody (Sigma-Aldrich).

Techniques: Expressing, Transformation Assay, Flow Cytometry