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: Biochemical pharmacology

Article Title: Biphasic modulation of cAMP levels by the contraceptive nomegestrol acetate. Impact on P-glycoprotein expression and activity in hepatic cells.

doi: 10.1016/j.bcp.2018.04.023

Figure Lengend Snippet: Fig. 1. NMGA increases P-gp expression and activity. a. Effect of NMGA on P-gp protein expression in HepG2 cells. Cells were treated with 0.5–500 nM NMGA or vehicle for 48 h and then subjected to western blot analysis. b. Effect of NMGA on P-gp protein expression in Huh7 cells. Cells were treated with 0.5–500 nM NMGA or vehicle for 48 h and then subjected to western blot analysis. Equal amounts of total protein (12 µg) were loaded in each lane. Representative blots for P-gp and GAPDH (loading control) are shown. Data are presented as % of control group (C) and expressed as mean ± standard deviation (n = 3). *Different from C, p < 0.05. c. Effect of NMGA on ABCB1 mRNA expression in HepG2 cells. Cells were treated with 5–500 nM NMGA or vehicle for 24 h. Then, ABCB1 mRNA expression was assessed by real time RT-PCR and normalized to the expression of HUPO, used as a housekeeping gene. Data are presented as % of control group (C) and expressed as mean ± standard deviation (n = 3). *Different from C, p < 0.05. d. Effect of NMGA on P-gp activity in HepG2 cells. Relative fluorescence due to calcein accu- mulation was quantified by flow cytometry in control (C) and NMGA treated cells (5, 50 and 500 nM) and inversely correlated with P-gp activity. Experiments were performed in the presence or absence of the selective P-gp inhibitor PSC833 (10 µM). Data are presented as % of C without PSC833 and expressed as mean ± standard deviation (n = 3). a: p < 0.05 vs C, b: p < 0.05 vs all other groups.

Article Snippet: 3-isobutyl-1-methylxanthine (IBMX), actinomycin D, calcein-AM, H-89 dihydrochloride (H89), KT5720, Rp-8-BrcAMP, mifepristone (RU 486), PSC833, Ro 20-1724 and SC68376 were from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Expressing, Activity Assay, Western Blot, Control, Standard Deviation, Quantitative RT-PCR, Cytometry

Journal: Acta pharmaceutica Sinica. B

Article Title: A mix & act liposomes of phospholipase A2-phosphatidylserine for acute brain detoxification by blood‒brain barrier selective-opening.

doi: 10.1016/j.apsb.2023.11.015

Figure Lengend Snippet: Figure 5 The investigation of factors influencing the interaction between POPS and PLA2. (AeC) The brain ARR when treated with PSC833, PLA2 inhibitor ACA, adenosine receptor inhibitor diphylline, and PLA2 inhibitor varespladib combined with PLA2. Data are presented as mean SD (n Z 10). (D) The brain ARR when treated with different inhibitors but without PLA2. Data are presented as mean SD (n Z 10). (EeP) Schematic diagrams of different treatment methods of PLA2 and POPS interaction with the cell (left) and fluorescent images of PI staining (right): (EeH) different mixing orders of PLA2 and POPS, (IeK) reaction components, (LeN) incubation temperatures, and (OeP) PLA2 in- hibitors. Scale bar Z 50 mm. **P < 0.01; ****P < 0.0001 vs. indicated. n.s., not significant.

Article Snippet: Component doses in the treatment groups were list as follows: phospholipid (80 mg/kg), HI-6 (30 mg/kg), PLA2 (250 U/kg), NECA (0.1 mg/kg), VEGF (2 mg/kg), LPS (2 mg/kg), borneol (50 mg/kg), mannitol (2 g/kg), Varespladib (50 mg/kg, MedChemExpress, Monmouth Junction, New Jersey, USA), diphyline (50 mg/kg, MedChemExpress, Monmouth Junction, New Jersey, USA), anthranilic acid (50 mg/kg, MedChemExpress, Monmouth Junction, New Jersey, USA), PSC833 (10 mg/kg, MedChemExpress, Monmouth Junction, New Jersey, USA), SCH58261 (5 mg/kg, MedChemExpress, Monmouth Junction, New Jersey, USA).

Techniques: Staining, Incubation

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

Journal: Neuropharmacology

Article Title: Differences in amyloid-β clearance across mouse and human blood-brain barrier models: Kinetic analysis and mechanistic modeling

doi: 10.1016/j.neuropharm.2014.01.023

Figure Lengend Snippet: Total brain clearance (BCITotal(%)) of 125I-Aβ40 in control (CNTRL), with P-gp inhibitor valspodar (VALSP) or LRP1 inhibitor RAP (A), BBB clearance (BCIBBB(%)) of 125I-Aβ40 in control and inhibitor groups (B), clearance of 125I-Aβ40 by brain degradation (BCIDegradation(%)) in control and inhibitor groups (C). Data represent mean±SEM for n=4; * P<0.05, ** P<0.01 and *** P<0.001. NS is not significant.

Article Snippet: To characterize role of P-gp and LRP1, 0.5 μl of ECF containing valspodar (40 μM; XenoTech, KS), a well-established P-gp inhibitor, or RAP (1 μM; Oxford Biomedical Research, MI), an LRP1 inhibitor, were intracerebrally administered 5 min prior to 125 I-Aβ 40 injection.

Techniques:

Journal: Neuropharmacology

Article Title: Differences in amyloid-β clearance across mouse and human blood-brain barrier models: Kinetic analysis and mechanistic modeling

doi: 10.1016/j.neuropharm.2014.01.023

Figure Lengend Snippet: A) In vitro uptake of intact 125I-Aβ40 by bEnd3 and hCMEC/D3 cells. Cellular level of intact 125I-Aβ40 is expressed in fmole/mg protein following cells treatment with 0.1 nM 125I-Aβ40 without (control; CNTRL) and with RAGE (RAGE-IgG), or P-gp (valspodar; VALSP), or LRP1 (RAP) inhibitors for 15 min and 12 h in bEnd3 and hCMEC/D3, respectively. B) Percent of degraded 125I-Aβ40 in the media of bEnd3 and hCMEC/D3 cells following treatment with 0.1 nM of 125I-Aβ40, with or without inhibitor. Data represent mean±SEM from three independent experiments; *P<0.05, **P< 0.01 and *** P<0.001.

Article Snippet: To characterize role of P-gp and LRP1, 0.5 μl of ECF containing valspodar (40 μM; XenoTech, KS), a well-established P-gp inhibitor, or RAP (1 μM; Oxford Biomedical Research, MI), an LRP1 inhibitor, were intracerebrally administered 5 min prior to 125 I-Aβ 40 injection.

Techniques: In Vitro