Journal: Journal of Veterinary Pharmacology and Therapeutics

Article Title: Comparison of Human and Canine P‐Glycoprotein Substrates at R, P, and H Binding Sites

doi: 10.1111/jvp.70034

Figure Lengend Snippet: Representative histograms representing flow cytometric analysis of a single well of either hMDR1 cells (A–C) or MDCKPGP cells (D–F) treated with the following fluorescent (“probe”) and non‐fluorescent P‐gp substrate combinations to confirm binding to their respective P‐gp binding sites: Rhodamine 123 and cyclosporine A for the R‐site; Hoechst 33342 and vinblastine for the H‐site; calcein AM and progesterone for the P‐site. Additional wells containing the negative control cisplatin and positive control PSC 833 were also evaluated. The x ‐axis represents fluorescence intensity, and the y ‐axis represents cell number. Experiments were run in triplicate and repeated on a separate day.

Article Snippet: The non‐P‐gp substrate cisplatin and the P‐gp substrates digoxin, cyclosporine A, ivermectin, loperamide, progesterone, PSC‐833, and vinblastine were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques: Binding Assay, Negative Control, Positive Control, Fluorescence

Journal: Journal of Veterinary Pharmacology and Therapeutics

Article Title: Comparison of Human and Canine P‐Glycoprotein Substrates at R, P, and H Binding Sites

doi: 10.1111/jvp.70034

Figure Lengend Snippet: Bar chart of (MFI [experimental drug + fluorescent P‐gp substrate]:MFI [fluorescent P‐gp substrate alone]) ratios for human ( x ‐axis) and canine ( y ‐axis) P‐gp at the R‐site (rhodamine 123, left panel) and P‐site (calcein AM, right panel) and standard deviations. Ratios of 1 indicate the compound is not a P‐gp substrate, ratios > 2 indicate the drug is a P‐gp substrate, and ratios > ~5 for calcein AM and > ~10 for rhodamine 123 indicate strong P‐gp substrate status. CIS, cisplatin (10 μM); CYC, cyclosporine A (1 μM); DIG, digoxin (1000 μM); IVM, ivermectin (800 μM); LOP, loperamide (10 μM); PRO, progesterone (10 μM); PSC, PSC 833 (1 μM); VIN, vincristine (50 μM). CIS and PSC are the negative and positive controls, respectively. Experiments were run in triplicate and repeated on a separate day. *Indicates statistically significant differences in MFI ratios between canine and human P‐gp.

Article Snippet: The non‐P‐gp substrate cisplatin and the P‐gp substrates digoxin, cyclosporine A, ivermectin, loperamide, progesterone, PSC‐833, and vinblastine were purchased from Tocris Biosciences (Minneapolis, MN).

Techniques:

Journal: Frontiers in Immunology

Article Title: P-glycoprotein expression skews mitochondrial dye measurements in T cells

doi: 10.3389/fimmu.2025.1560104

Figure Lengend Snippet: P-glycoprotein (P-gp) expression affects mitochondrial dye measurements in T cells. (a, b) MTG signals from splenic naïve (CD44 - CD62L + ) and memory (CD44 + CD62L + ) CD8 T cells stained with or without PSC833 (1uM). (c) Protein expression level of P-glycoprotein based on Variance Stabilization Normalization (VSN) Normalized Intensities from proteomic analysis. (d, e) MTG and (f, g) TMRE signals from thymic CD4 T (CD8a - CD1d-aGC-tetramer - CD4 + TCRβ hi ) and iNKT (CD8a - CD1d-aGC-tetramer + TCRb int ) cells stained with or without PSC833 as in (a) . (h, i) MTG and (j, k) TMRE signals from PBMC CD4 T (CD8a - CD1d-aGC-tetramer - CD4 + TCRb hi ) and iNKT (CD8a - CD1d-aGC-tetramer + TCRb int ) cells stained with or without PSC833 as in (a) . The data shown in (a, b, d-k) are one representative experiment out of three independent experiments. The data shown in c are from samples collected from two sorting experiments and proteomes analyzed together. ns, non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, paired Student’s t test.

Article Snippet: PSC833 was from Santa Cruz Biotechnology.

Techniques: Expressing, Staining

Journal: Biomaterials

Article Title: A bioprinted and scalable model of human tubulo-interstitial kidney fibrosis.

doi: 10.1016/j.biomaterials.2024.123009

Figure Lengend Snippet: Fig. 1. Generation of stable human kidney cell lines with proven origin. A: Schematic of the human kidney and the cortex illustrates the source of isolated cells. B: Immunofluorescence staining of PDGFRβ+, CD10+ and CD31+ specific antibodies for cells in cortex of human kidney. C: Enumeration of the workflow from isolating the cells to the generation of genetically tagged cells. D: Representative pictures of genetically labeled immortalized PDGFRβ+, proximal tubular epithelial and endothelial cell lines. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. E: Principal component analysis (PCA) plot of the generated three cell lines. The three cell lines show higher variability from each other, while technical replicates are similar to each other. F: Shared DE genes between isolated cell lines and human bulk RNA seq [13]. G: CD10+ (red) cells stained for cell-specific proximal tubule epithelial markers cubilin (green, left) and CD13 (green, right) including DAPI. H: Heatmap of bulk RNA-sequencing data demonstrating the genes expressed in CD10+. I: P-gp Calcein-AM transporter assay of CD10+ and negative control PDGFRβ+ cells, with or without transporter inhibitor PSC833. ***p < 0.001 (one-way ANOVA analysis followed by Tukey post-test), n = 3 with each 12 replicates. J: Presence of intracellular vWF (red) in CD31+ (green) cells including DAPI. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were treated for 1 h with 5 μM P-gp transporter inhibitor PSC833 (Tocris Biosciences, Bristol, UK) in KH buffer at 37 ◦C, 5 % (v/v) CO2, and afterwards exposed for 1 h to 1 μM Calcein-AM (Life Technologies Europe BV) with or without 5 μM PSC833 in KH buffer.

Techniques: Isolation, Immunofluorescence, Staining, Labeling, Generated, RNA Sequencing, Negative Control