mrp5 rat  (OriGene)

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Primers used for PCR (5′-3′).

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques:

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Functional overexpression of MRP5 in human pancreatic cancer MIA Paca-2 and PANC-1 cells. (A) High MRP5 mRNA expression in both MIA PaCa-2 and PANC-1 cells (in red colour) compared to other pancreatic cancer cell lines from the Wagner dataset stored in ONCOMINE ( https://www.oncomine.org ). (B and C) MRP5 protein detected in representative flow cytometry histogram of cell surface staining using the anti-MRP5 primary antibody (red) and isotype control IgG2a (green) on (B) MIA Paca-2 and (C) PANC-1 cells. Both the primary antibody and isotype control were labelled with Alexa Fluor 488 secondary antibody. The x-axis is the fluorescence signal intensity displayed in a linear log scale. (D) Functional expression of MRP5 detected by BCECF accumulation in MIA Paca-2 and PANC-1 cells at 15 min in the presence and absence of 50 µM benzbromarone. All data are normalised to the fluorescence intensity determined in the absence of benzbromarone. The bars represent the mean and standard deviation from three independent experiments performed in triplicates. ****P<0.0001 according to Sidak's post-hoc test that followed two-way ANOVA. MRP5, multidrug resistance protein 5.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Functional Assay, Over Expression, Expressing, Flow Cytometry, Staining, Fluorescence, Standard Deviation

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: MRP5 expression at the mRNA level in (A) MIA Paca-2 and (B) PANC-1 clones transduced with scrambled control and multidrug resistance protein 5-short-hairpin RNA. Relative MRP5 mRNA expression was detected by reverse transcription-quantitative PCR. MRP5 mRNA expression was normalised to the reference gene GAPDH and relative quantitation of gene expression was calculated using the comparative threshold cycle method (2 −ΔΔCq ). All data were expressed as the mean and standard deviation from three independent experiments performed in duplicates. Cell surface protein expression of MRP5 in (C) MIA Paca-2 and (D) PANC-1 cells presented as the mean percentage of the scrambled control. The bar represents the mean and standard deviation from three independent experiments performed in triplicates. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001 from Dunnett's post-hoc test that followed one-way ANOVA for comparison of all MRP5 knockdown clones to the scrambled control. (E and F) MRP5 protein detected in representative flow cytometry histograms of cell surface staining using anti-MRP5 primary antibody and Alexa Fluor 488 secondary antibody on (E) MIA Paca-2 and (F) PANC-1 clones. The x-axis is the fluorescence signal intensity displayed in a linear log scale. MRP5, multidrug resistance protein 5; shRNA, short-hairpin RNA.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Expressing, Clone Assay, Transduction, shRNA, Real-time Polymerase Chain Reaction, Quantitation Assay, Standard Deviation, Comparison, Flow Cytometry, Staining, Fluorescence

Journal: Oncology Reports

Article Title: Multidrug resistance protein 5 affects cell proliferation, migration and gemcitabine sensitivity in pancreatic cancer MIA Paca‑2 and PANC‑1 cells

doi: 10.3892/or.2023.8666

Figure Lengend Snippet: Ligands docked on MRP5 macromolecule. Binding of (A) gemcitabine and (B) cyclic guanosine monophosphate with MRP5. MRP5 is depicted in red colour in ribbon form. MRP5, multidrug resistance protein 5.

Article Snippet: After fixation in 1% paraformaldehyde (PFA) (cat. no. P6148; Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, the cells underwent permeabilization in 0.2% saponin (cat. no. 47036-50G-F, Sigma-Aldrich; Merck KGaA) at 4°C for 15 min, blocking in 5% bovine serum albumin (BSA) (cat. no. NZ21-69100-038; pH Scientific) at room temperature for 15 min, and staining with anti-MRP5 rat monoclonal antibody (M5II-54) (1:20 in 2% BSA; cat. no. MA1-35684; Thermo Fisher Scientific, Inc.) or Rat IgG2α Isotype Control primary antibody (1:20 in 2% BSA; cat. no. 02-9688, Thermo Fisher Scientific, Inc.) at 4°C for 1 h. The cells were then stained with goat anti-Rat IgG H&L (Alexa Fluor ® 488) secondary antibody (1:200 in 2% BSA; cat. no. ab96887; Abcam) at 4°C for 1 h. The cells were washed three times with PBS containing 0.1% NaN 3 and 0.1% saponin between each step.

Techniques: Binding Assay