third-generation lentiviral vector particles  (Cell Biolabs Inc)

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 1. Expression of T2R14 in human pancreatic tissue. (A) T2R14 mRNA transcripts from 75 patients with PDAC derived from the HIPO databank (median, 5.8 rpkm). Data are presented as quantified transcript levels in rpkm. (B) Kaplan‑Meier univariate survival analysis comparing patients with PDAC and high (≥5.8 rpkm; n=38) or low (<5.8 rpkm; n=37) T2R14 expression levels of T2R14. The median T2R14 expression level was used as the cut‑off. (C) Representative images of T2R14 staining (brown) in normal pancreas, low grade PanIN, high grade PanIN, PDAC from the periphery and the center, and lymph node metastasis tissue specimens. (D) Allred scoring system was used to semi‑quantify T2R14 protein expression in 102 PDAC tumor samples. Data are presented as the median ± IQR (E) Distribution of T2R14 protein expression intensity in PDAC tumors. Samples were categorized according to the Allred scoring system: Negative, score 0; low, score 2‑3; medium, score 4‑6; high, score 7‑8. Bars represent the proportion of PDAC tumors with respective Allred scores. HIPO, Heidelberg Institute of Personalized Oncology; PanIN, pancreatic intraepithelial neoplasia; PDAC, pancreatic ductal adenocarcinoma; rpkm, reads per kilobase million; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, Derivative Assay, Staining

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 2. Expression of T2R14 in human pancreatic cell lines. (A) mRNA expression levels of T2R14 were analyzed in various pancreatic cancer cell lines by reverse transcription‑quantitative polymerase chain reaction and were normalized to HPRT mRNA levels. SU.86.86 cells exhibited the highest expression levels. The MCF‑7 breast cancer cell line served as a positive control. Data are presented as the mean ± SD of three independent experiments. (B) Flow cytometry was used to detect T2R14 surface protein expression in pancreatic cell lines. The MCF‑7 breast cancer cell line served as a positive control. Solid lines represent T2R14 antibody binding and the dotted lines represent the IgG isotype control. (C) Quantification of T2R14 protein expression as the mean fluo‑ rescent intensity normalized to mode. Out of the pancreatic cancer cell lines, the highest T2R14 protein expression levels were found in SU.86.86 and PANC‑1 cells. The MCF‑7 breast cancer cell line served as a positive control. Data are presented as the mean ± SD of three independent experiments. (D) Representative confocal images showing human pancreatic tumor cells exhibiting immunoreactivity to T2R14 (green staining). Nuclear DNA was stained with DAPI (blue) and F‑actin with Texas Red™‑X Phalloidin (red). (E) Representative images of IgG isotype staining used as negative control to exclude non‑specific T2R14 antibody binding. (F) High magnification images of immunofluorescence staining in SU.86.86 and PANC‑1 cells demonstrating robust expression of T2R14 on the cell membrane and in the cytoplasm. MFI, mean fluorescence intensity; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, Polymerase Chain Reaction, Positive Control, Flow Cytometry, Binding Assay, Control, Staining, Negative Control, Immunofluorescence, Membrane, Fluorescence

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 4. Efficacy of shRNA‑mediated silencing of T2R14. (A and E) Reverse transcription‑quantitative polymerase chain reaction was used to assess T2R14 mRNA expressions levels in cells transduced with T2R14‑shRNA and control‑shRNA. The expression levels of cells transduced with control‑shRNA was set at 100%. T2R14 mRNA was reduced in T2R14‑shRNA‑transduced PANC‑1 and SU.86.86 cells (three independent experiments are shown, of which two data points are overlaid and displayed as single points in the graph). (B and F) Cell surface expression of T2R14 protein in PDAC cells transduced with control‑shRNA (light grey) or T2R14‑shRNA (dark grey) was measured by flow cytometry. Solid lines represent specific T2R14‑antibody signal and dotted/dashed lines represent the isotype control. (C and G) Quantification of MFI showed a reduction in T2R14 expression in PANC‑1 and SU.86.86 cells following shRNA transduction. The MFI for cells transduced with control‑shRNA was set at 100%. Three independent experiments are shown. Two data points are overlaid and thus displayed as single points in the graph. (D and H) Representative images of immunofluorescence (red staining) in cells transduced with T2R14‑shRNA and control‑shRNA. Reduced immunofluorescence staining in silenced cells confirming a sufficient knockdown of T2R14 at the protein level. MFI, mean fluorescence intensity; shRNA, short hairpin RNA; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: Polymerase Chain Reaction, Transduction, Expressing, Flow Cytometry, Control, shRNA, Immunofluorescence, Staining, Knockdown, Fluorescence

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 5. Cytotoxic effects of apigenin are independent of T2R14 expression. (A) SU.86.86 and (B) PANC‑1 cells were exposed to increasing concentrations of apigenin. Cell viability was assessed using the MTS assay. Cells in the T2R14‑shRNA group were compared with those in the control‑shRNA group. All values were normalized to the group of cells treated with PBS/DMSO only. Data are presented as the mean ± SD of three independent experiments. shRNA, short hairpin RNA; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: Expressing, MTS Assay, shRNA

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 7. Apigenin does not affect the cytotoxicity of FOLFIRINOX, 5‑FU or Gem in PANC‑1 cells. (A‑C) PANC‑1 cells were exposed to a range of concentra‑ tions of chemotherapeutic treatments (Gem, 5‑FU and FOLFIRINOX), in the presence or absence of 10 µM apigenin. (D‑F) T2R14‑shRNA‑transfected cells were exposed to a range of concentrations of chemotherapeutic treatments (Gem, 5‑FU and FOLFIRINOX), in the presence or absence of 10 µM apigenin. MTS assays were applied for the determination of cell viability. Values were normalized to untreated cells. Data are presented as the mean ± SD of at least three independent experiments, each of them performed in triplicate. Statistical significance was assessed using the extra sum‑of‑squares F test. 5‑FU, 5‑fluorouracil; FOLFIRINOX, 5‑FU, leucovorin, irinotecan and oxaliplatin; Gem, gemcitabine; shRNA, short hairpin RNA; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: shRNA

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 6. Apigenin synergistically enhances the cytotoxicity of FOLFIRINOX and 5‑FU in SU.86.86 cells. (A‑C) SU.86.86 cells were exposed to a range of concentrations of chemotherapeutic treatments (Gem, 5‑FU and FOLFIRINOX), in the presence or absence of 10 µM apigenin. (D‑F) T2R14‑shRNA‑transfected cells were exposed to a range of concentrations of chemotherapeutic treatments (Gem, 5‑FU and FOLFIRINOX), in the presence or absence of 10 µM apigenin. MTS assays were applied for the determination of cell viability. Values were normalized to untreated cells. Data are presented as the mean ± SD of at least three independent experiments, each of them performed in triplicate. Statistical significance was assessed using the extra sum‑of‑squares F test. 5‑FU, 5‑fluorouracil; FOLFIRINOX, 5‑FU, leucovorin, irinotecan and oxaliplatin; Gem, gemcitabine; shRNA, short hairpin RNA; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: shRNA

Journal: International journal of oncology

Article Title: Potential role of the bitter taste receptor T2R14 in the prolonged survival and enhanced chemoresponsiveness induced by apigenin.

doi: 10.3892/ijo.2022.5454

Figure Lengend Snippet: Figure 8. API inhibits the migration of PANC‑1 and SU.86.86 cells in a concentration‑dependent manner. (A and D) Migratory capacity of SU.86.86 (upper panel) and PANC‑1 (lower panel) cells. (B and E) Migratory capacity of T2R14‑shRNA cells. Migration of control cells treated with PBS only was set as 100%. Semi‑quantification of the relative numbers of migrated cells exhibited a gradual decrease in migration with increasing API concentration in both cell lines with silenced T2R14 and control cells. The number of migrated cells was normalized to the untreated cell groups. Data are presented as the mean ± SEM of three independent experiments. Statistical significance was assessed using one‑way ANOVA and Bonferroni's multiple comparison test. ***P<0.001: ****P<0.0001. (C and F) Representative images of the migrated SU.86.86 and PANC‑1 cells stained with crystal violet. Cells were treated with three concentrations of API. Relative migration decreased with rising apigenin concentration. Magnification, x200. API, apigenin; shRNA, short hairpin RNA; T2R14, bitter taste receptor 14.

Article Snippet: Third generation T2R14 pGFP‐C‐shLenti shRNA vectors (cat. no. 50840) and non‐targeting pGFP‐C‐shLenti shRNA control vectors (cat. no. TR30021) were purchased from OriGene Technologies, Inc.

Techniques: Migration, Control, Concentration Assay, Comparison, Staining, shRNA