Journal: PLOS One

Article Title: A VPS33B CRISPR knockout study: In vitro evidence of an adhesion defect

doi: 10.1371/journal.pone.0343240

Figure Lengend Snippet: A) Initial PCR reaction and subsequent T7EI assay carried out on gDNA collected from plasmid h1 transfected (left), plasmid h2 (middle), or untransfected (NT; right) RPTEC-TERT1 cells. B) Western blot (n = 4) analysis of VPS33B expression in clones of a human proximal tubule cell line (RPTEC-TERT1). Data represents mean ± SEM. ****P < 0.0001. ANOVA. Tukey’s multiple comparisons test. C) Sequence of 5’ of the first exon of the VPS33B gene and the deletions (compound heterozygotes – blue, homozygotes – red) identified in CRISPR-treated RPTEC-TERT1 cells. Exon 1 and the CRISPR single-guide RNAs (sgRNAs) were depicted as a blue rectangle and green labels, respectively. D) Predicted changes to the amino acid sequence of VPS33B resulting from the mutations in B. Red dashes represent amino acid deletions and red line represents frameshift site.

Article Snippet: RPTEC-TERT1 cells (ATCC, #CRL-4031TM) were cultured in DMEM:F12 Medium (ATCC® 30–2006TM), supplemented with hTERT RPTEC Growth Kit (ATCC® ACS-4007TM) (1% Supplement A and 1.6% Supplement B), 2% FBS, and 0.1 mg/mL GeneticinTM Selective Antibiotic (G418 Sulfate) (Thermo Fisher, #10131035).

Techniques: T7EI Assay, Plasmid Preparation, Transfection, Western Blot, Expressing, Clone Assay, Sequencing, CRISPR

Journal: PLOS One

Article Title: A VPS33B CRISPR knockout study: In vitro evidence of an adhesion defect

doi: 10.1371/journal.pone.0343240

Figure Lengend Snippet: x10 brightfield images of A) Control, B) VPS33B-KO clone 1, and C) VPS33B-KO clone 2 RPTEC-TERT1 cells in 96-well plates after 1, 3, 5, 7, and 10 days of culture. Blue arrows: elongated RPTEC-TERT1 cells. Black arrow: Monolayer peeling. Scale bar: 100 µm. D) Survival analysis for time until monolayer peeling (n = 7). Log-rank test for trend. p < 0.0001. E) Analysis of absorbance of crystal violet one day after seeding. ANOVA. Tukey’s multiple comparisons test (n = 5). F) Results of a cell detachment assay carried out on Control and VPS33B-KO RPTEC-TERT1 cells. Two-way ANOVA. Tukey’s multiple comparisons test. Control vs KO clone 1: #p < 0.05; ###p < 0.001, ####p < 0.0001. Control vs KO clone 2: *p < 0.05; ***p < 0.001, ****p < 0.0001 (n = 5). Representative x40 images of G) Control: CRISPR-transfected control, H) KO1: VPS33B-KO RPTEC-TERT1 Clone 1, I) KO2: VPS33B-KO RPTEC-TERT1 Clone 2 stained with DAPI (blue), anti-phospho-paxillin antibody (yellow), and phalloidin-stained F-actin (magenta). Cyan arrows indicate linear junctional staining of phospho-paxillin. White arrows indicate nuclear phospho-paxillin staining. n = 3. Scale bar = 50 µm. Analysis of J) mean size of phospho-paxillin-positive puncta area, K) number of punctae per cell, and L) association of phospho-paxillin-positive punctae with the actin cytoskeleton comparing Control cells to a pooled sample of VPS33B-KO clones. n = 3 per genotype. T-test. Data represents mean ± SEM. *p < 0.05. Abbreviations: a.u.: arbitrary units.

Article Snippet: RPTEC-TERT1 cells (ATCC, #CRL-4031TM) were cultured in DMEM:F12 Medium (ATCC® 30–2006TM), supplemented with hTERT RPTEC Growth Kit (ATCC® ACS-4007TM) (1% Supplement A and 1.6% Supplement B), 2% FBS, and 0.1 mg/mL GeneticinTM Selective Antibiotic (G418 Sulfate) (Thermo Fisher, #10131035).

Techniques: Control, CRISPR, Transfection, Staining, Clone Assay

Journal: Toxicology

Article Title: An in vitro-in silico workflow for predicting renal clearance of environmental chemicals and drugs

doi: 10.1016/j.tox.2025.154336

Figure Lengend Snippet: Schematic overview of experimental design and modeling workflow. hTERT-RPTEC and hTERT-RPTEC-OAT1 cells were used in 96-well plates and Transwells to measure compound concentrations in cell lysates (24 h only) and media (4 and 24 h). Data were integrated into two- and three-compartment models to estimate media-to-cell ratios and transport dynamics. Predicted renal clearance values were then compared with reported human in vivo data.

Article Snippet: These cells were cultured as recommended by the vendor in DMEM/F12 (#30–2006, ATCC) supplemented with the “hTERT Immortalized RPTEC Growth Kit” (#ACS-4007, ATCC), with a final concentration of 0.1 mg/mL Geneticin TM Selective Antibiotic (G418 Sulfate; Gibco, Waltham, VA).

Techniques: In Vivo

Journal: Toxicology

Article Title: An in vitro-in silico workflow for predicting renal clearance of environmental chemicals and drugs

doi: 10.1016/j.tox.2025.154336

Figure Lengend Snippet: Cytotoxicity and barrier integrity in RPTEC cultures following compound treatment. (A) Baseline LDH activity and barrier integrity of vehicles prior to treatment. (B) LDH release (reported as % of vehicle) after 24 h treatment with PFAS and reference compounds across plate and Transwell configurations. TAB (positive control) induced marked cytotoxicity, while most treatments showed no significant increase compared to vehicle. See for the data. (C) TEER (% of pre-treatment) in Transwells demonstrated maintained barrier function under most conditions, with significant decreases observed for the TAB positive control and for selected PFAS when added basolateral-to-apical (B→A). Asterisks denote significance (p < 0.05) by one-way ANOVA with Dunnett’s post hoc test. Box-and-whisker plots are shown with the box showing the inter-quartile range and whiskers = min–max (n ≥ 3 for 96-well plate studies, n ≥ 4 for Transwell studies).

Article Snippet: These cells were cultured as recommended by the vendor in DMEM/F12 (#30–2006, ATCC) supplemented with the “hTERT Immortalized RPTEC Growth Kit” (#ACS-4007, ATCC), with a final concentration of 0.1 mg/mL Geneticin TM Selective Antibiotic (G418 Sulfate; Gibco, Waltham, VA).

Techniques: Activity Assay, Positive Control, Whisker Assay

Journal: Toxicology

Article Title: An in vitro-in silico workflow for predicting renal clearance of environmental chemicals and drugs

doi: 10.1016/j.tox.2025.154336

Figure Lengend Snippet: Compound recovery in RPTEC cultures across platforms. Amounts of each test compound recovered (pmoles) after 24 h treatment in (A) hTERT-RPTEC (96-well plates), (B) hTERT-RPTEC-OAT1 (96-well plates), (C) hTERT-RPTEC-OAT1 Transwells dosed apical-to-basolateral (A→B), and (D) hTERT-RPTEC-OAT1 Transwells dosed basolateral-to-apical (B→A). Recovery was measured from donor media (white), cell lysates (orange), and recipient media (purple). Results highlight differences in distribution between compartments and across culture formats. Box-and-whisker plots are shown with the box showing the inter-quartile range and whiskers = min–max (n ≥ 3 for 96-well plate studies, n ≥ 4 for Transwell studies).

Article Snippet: These cells were cultured as recommended by the vendor in DMEM/F12 (#30–2006, ATCC) supplemented with the “hTERT Immortalized RPTEC Growth Kit” (#ACS-4007, ATCC), with a final concentration of 0.1 mg/mL Geneticin TM Selective Antibiotic (G418 Sulfate; Gibco, Waltham, VA).

Techniques: Whisker Assay

Journal: Toxicology

Article Title: An in vitro-in silico workflow for predicting renal clearance of environmental chemicals and drugs

doi: 10.1016/j.tox.2025.154336

Figure Lengend Snippet: Donor to recipient ratios across culture formats. (A) Ratio of cell (lysate) to media concentrations after 24 h exposure in hTERT-RPTEC (Parent) and hTERTRPTEC-OAT1 (OAT1) cultured in 96-well plates. (B) Cell to media (donor) ratios in hTERT-RPTEC-OAT1 Transwells following apical-to-basolateral (A→B) or basolateral-to-apical (B→A) dosing. (C) Ratio of recipient to donor media concentrations in Transwells for both dosing orientations. The red dashed line denotes equilibration (equal concentration in donor and recipient media). Values to the right of the line indicate higher levels in the recipient compartment (cells or recipient media), suggesting selective transport, while values to the left indicate retention in the donor compartment, consistent with barrier function or limited transport.

Article Snippet: These cells were cultured as recommended by the vendor in DMEM/F12 (#30–2006, ATCC) supplemented with the “hTERT Immortalized RPTEC Growth Kit” (#ACS-4007, ATCC), with a final concentration of 0.1 mg/mL Geneticin TM Selective Antibiotic (G418 Sulfate; Gibco, Waltham, VA).

Techniques: Cell Culture, Concentration Assay

Journal: Toxicology

Article Title: An in vitro-in silico workflow for predicting renal clearance of environmental chemicals and drugs

doi: 10.1016/j.tox.2025.154336

Figure Lengend Snippet: Comparison of predicted (this study) and observed (published) human renal clearance values. Predicted total renal clearance (CL renal,total ) from in vitro – in silico models was compared with reported in vivo values for PFAS (black circles) and pharmaceutical compounds (purple squares). (A) Predictions from 96-well plate studies using two-compartment models with hTERT-RPTEC (filled icons) and hTERT-RPTEC-OAT1 cells (open icons). (B) Predictions from hTERT-RPTEC-OAT1 Transwell assays using three-compartment modeling (left) or two-compartment modeling for apical-to-basolateral (middle) and basolateral-to-apical (right) dosing. (C) Same as (A) for the validation experiment. Dashed black lines represent regression fits, and the red dotted line indicates perfect agreement. Pearson (r) and Spearman (ρ) correlation coefficients are shown for PFAS (black) and pharmaceutical compounds (purple). See for the data.

Article Snippet: These cells were cultured as recommended by the vendor in DMEM/F12 (#30–2006, ATCC) supplemented with the “hTERT Immortalized RPTEC Growth Kit” (#ACS-4007, ATCC), with a final concentration of 0.1 mg/mL Geneticin TM Selective Antibiotic (G418 Sulfate; Gibco, Waltham, VA).

Techniques: Comparison, In Vitro, In Silico, In Vivo, Biomarker Discovery