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: International journal of molecular sciences

Article Title: N-Acetylcysteine Attenuates Aβ-Mediated Oxidative Stress, Blood-Brain Barrier Leakage, and Renal Dysfunction in 5xFAD Mice.

doi: 10.3390/ijms26094352

Figure Lengend Snippet: Figure 3. Effect of NAC on blood–brain barrier integrity in 5xFAD and WT mice. (A) Brain capillary 4-HNE levels (µg/mg protein) in untreated 5xFAD mice compared to those in untreated WT mice (p = 0.0018) or NAC-treated 5xFAD mice (p = 0.0333). Circles represent technical replicates. Data are presented as mean (filled columns) ± standard error of the mean (SEM). (B) Isolated capillaries were incubated with 2 µM NBD-CSA, a fluorescent P-glycoprotein-specific substrate, for 1 h alone or with PSC833. Specific luminal NBD-CSA fluorescence was taken as the difference between total luminal fluorescence and fluorescence in the presence of the P-gp inhibitor PSC833. Data are mean ± SEM for 10 capillaries from one preparation (pooled tissue from 15 mice per group), in arbitrary fluorescence units (scale 0–255). **, significantly lower than control, p < 0.01. (C) Plasma S100β levels (pg/mL) were 2.5-fold higher (p < 0.0001) for untreated 5xFAD mice (red column; n = 15 mice) than for untreated WT mice (blue column; n = 15 mice). NAC treatment (green column; n = 15 mice) significantly lowered S100β levels in 5xFAD mice (p < 0.0001). Circles represent biological replicates. (D) Texas Red leakage from capillary lumens was imaged over time using a confocal microscope for untreated WT mice (blue line), untreated 5xFAD mice (red line), and NAC-treated 5xFAD mice (green line). 100 mM mannitol served as a positive control for barrier opening (black line). Data are mean ± SEM for 7 brain capillaries per time point from one capillary isolation per group from 15 mice. Shown are arbitrary units (0–255). First-order efflux rates were calculated using non-linear regression.

Article Snippet: PSC833 was a kind gift from Novartis (Basel, Switzerland).

Techniques: Isolation, Incubation, Fluorescence, Control, Clinical Proteomics, Microscopy, Positive Control

Journal: Biomedical Reports

Article Title: Scutellarein enhances cisplatin‑induced apoptotic effects by suppressing the PI3K/AKT‑MDR1 pathway in human NPC/HK1 nasopharyngeal carcinoma cells

doi: 10.3892/br.2025.1938

Figure Lengend Snippet: Scutellarein enhances cisplatin-induced inhibition of cell viability and release of cytokeratin 18 fragments by inhibiting the PI3K/AKT-MDR1 pathway in NPC/HK1 cells. (A) NPC/HK1 cells were treated without (Ctrl) or with 12.5 µM scutellarein, 4.15 µM cisplatin, or their combination for 48 h. Protein expression of p-AKT, AKT, MDR1, and GAPDH was examined using an immunoblotting assay. (B) NPC/HK1 cells were treated without (Ctrl) or with 4.15 µM cisplatin, or 4.15 µM cisplatin plus 10 µM LY294002 for 48 h. Upper panel, protein expression of p-AKT, AKT, MDR1, and GAPDH was examined using an immunoblotting assay. Lower panel, cytokeratin 18 fragment levels in the cell culture supernatant were measured by ELISA. (C) NPC/HK1 cells were treated without (Ctrl) or with 4.15 µM cisplatin, or 4.15 µM cisplatin plus 10 µM PSC833 for 48 h. Upper panel, protein expression of MDR1 was examined using an immunoblotting assay. Middle panel, cell viability was assessed using the MTT assay. Lower panel, cytokeratin 18 fragment levels in the cell culture supernatant were measured by ELISA. Statistical significance was indicated as follows: * P<0.05 vs. Ctrl; and # P<0.05 vs. Cis alone; n=3. p-, phosphorylated; MDR1, multidrug resistance protein 1; Ctrl, control; Scu, scutellarein; Cis, cisplatin; LY, LY294002.

Article Snippet: Scutellarein, cisplatin, dimethyl sulfoxide (DMSO), 3-methyladenine (3-MA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), PSC833, and LY294002 were obtained from MilliporeSigma.

Techniques: Inhibition, Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, MTT Assay, Control

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