Journal: bioRxiv

Article Title: 3D brain angiogenesis model to reconstitute maturation of functional human blood-brain barrier in vitro

doi: 10.1101/471334

Figure Lengend Snippet: (A) 3D confocal image of p-gp protein (red) expressed in tri-cultured vasculature at day 7. Right section view is zoomed from left 3D projection image which describes intraluminal oriented expression of p-gp (pointed as yellow arrow heads). ECs and Nucleus were labeled by anti-CD31 (green) and Hoeschst 33342 (blue), respectively. Scale bar: 20μm. (B) Conceptual description of calcein-AM efflux assay (bottom) which mimics actual effect of p-gp inhibitor on drug resistance by p-gp in vivo (top). (C) Schematic description of work flow for calcein-AM efflux assay in developed in vitro BBB with or without p-gp inhibitor Valsopodar. (D) Representative image of calcein fluorescence in each time point. Vasculature was cultured in EC mono-culture or BBB tri-culture condition with or without p-gp inhibitor pre-treatment. Scale bar: 100μm. (E) Change of calcein fluorescence intensity at the designated region on vessel wall (yellow boxes in 0hour images) every 120 minutes. (F) Quantitative comparison of change in calcein fluorescence intensity during total experiment duration (640 minutes) normalized by initial intensity. (E, F) n=65 for EC only/DMSO Control; n=70 for EC only/Valsopodar treated; n=50 for BBB Tri-culture/DMSO control; n=110 for BBB Tri-culture/Valsopodar treated. Error bars represent SEM. Statistical comparisons between analyzed values of DMSO control and Valsopodar treated group in each time point was obtained from unpaired two-tailed Student’s t-test, with the p value threshold for statistical significance set at *p<0.05; **p<0.005; ***p<0.00001.

Article Snippet: A fully perfused vascular network of EC monoculture or BBB tri-culture was pre-treated with the p-gp inhibitor Valsopodar (PSC-833; Adooq) for 10 hours at a concentration of 10μM.

Techniques: Cell Culture, Expressing, Labeling, In Vivo, In Vitro, Fluorescence, Two Tailed Test

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Expression and function of MDR1 transporter in RPTEC cells. (A) Protein expression of MDR1 in RPTEC cells was detected by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period) and then treated with the culture media with or without PSC833 (2 μM) for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. Data are presented as mean ± SE (n = 4). Asterisk (*) represents statistically significant differences (p < 0.05) compared with RPTEC cells treated only with rhodamine 123 (no PSC833).

Article Snippet: PSC833 was purchased from XenoTech, LLC (Lenexa, KS).

Techniques: Expressing, Western Blot, Fluorescence

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with paraquat (100 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). (A) Intracellular paraquat accumulation was quantified by ELISA normalized to protein concentrations of the cellular lysates. (B) Paraquat concentrations in the culture media were quantified by ELISA and compared with levels detected in RPTEC cell lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated only with paraquat (no PSC833).

Article Snippet: PSC833 was purchased from XenoTech, LLC (Lenexa, KS).

Techniques: Inhibition, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Paraquat accumulation in RPTEC cells following siRNA knockdown of MDR1. RPTEC cells were transfected with siRNA duplexes targeted against human MDR1. (A) Protein expression of MDR1 at 72 h was assessed by Western blot analysis. (B) RPTEC cells were treated with rhodamine 123 (25 μM) in the presence or absence of the MDR1 inhibitor, PSC833 (2 μM), for 30 min (uptake period), washed, and then incubated in fresh culture media with or without PSC833 (2 μM) for 60 min (efflux period). MDR1 siRNA transfected RPTEC cells were treated with rhodamine (25 μM) for 30 min (uptake period), washed, and then incubated in fresh culture media for 60 min (efflux period). Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) RPTEC cells were treated as described in (B) using paraquat (100 μM) as a substrate. Intracellular paraquat accumulation was quantified by ELISA and normalized to protein concentrations of the cellular lysates. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells without PSC833 or MDR1 siRNA.

Article Snippet: PSC833 was purchased from XenoTech, LLC (Lenexa, KS).

Techniques: Knockdown, Transfection, Expressing, Western Blot, Incubation, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Toxicological Sciences

Article Title: MDR1 Transporter Protects Against Paraquat-Induced Toxicity in Human and Mouse Proximal Tubule Cells

doi: 10.1093/toxsci/kfu141

Figure Lengend Snippet: Cytotoxicity of paraquat in RPTEC cells following MDR1 inhibition. RPTEC cells were treated with different concentrations of paraquat (0–25 mM) for 3 h in the presence and absence of the MDR1 inhibitor PSC833 (2 μM) (uptake period) and then incubated in fresh culture media with or without PSC833 for 69 h (efflux period). (A) Cytotoxicity was assessed using the LDH assay and expressed as percent of LDH released into the cell culture media relative to total LDH activity. (B) RPTEC cells were pretreated with vehicle or PSC833 (2 μM) for 2 h and then treated with vehicle or paraquat (100 μM) for 24 h. Protein expression of Ho-1 was semi-quantified by Western blot. β-actin was used as a loading control. Data are presented as mean ± SE (n = 3). Asterisks (*) represent statistically significant differences (p < 0.05) compared with RPTEC cells treated by paraquat (no PSC833).

Article Snippet: PSC833 was purchased from XenoTech, LLC (Lenexa, KS).

Techniques: Inhibition, Incubation, Lactate Dehydrogenase Assay, Cell Culture, Activity Assay, Expressing, Western Blot, Control