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 MDR1-transfected HEK293 cells. HEK293 cells were stably transfected with empty vector (EV) or full-length human MDR1 plasmids. (A) Protein expression of MDR1 was detected by Western blot analysis. β-actin was used as a loading control. HEK-EV and MDR1 transfected cells were treated with rhodamine 123 (25 μM) or paraquat (100 μM) for 30 min (uptake period), washed, and then incubated in fresh culture medium for 60 min (efflux period). (B) Intracellular fluorescence of rhodamine 123 was detected using a Nexcelom Cellometer Vision and expressed as relative fluorescence units. (C) Intracellular paraquat accumulation was quantified by ELISA and normalized to protein lysate concentrations. Data are presented as mean ± SE (n = 4). Asterisks (*) represent statistically significant differences (p < 0.05) compared with HEK-EV cells.

Article Snippet: Following washing and centrifugation, the cell pellet was suspended in PBS and the intensity of fluorescence for each cell was quantified on a Cellometer Vision cell counter using filter cube VB-595–502 (excitation/emission: 525/595 nm) (Nexcelom Bioscience LLC, Lawrence, MA).

Techniques: Transfection, Stable Transfection, Plasmid Preparation, Expressing, Western Blot, Control, 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: 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: Following washing and centrifugation, the cell pellet was suspended in PBS and the intensity of fluorescence for each cell was quantified on a Cellometer Vision cell counter using filter cube VB-595–502 (excitation/emission: 525/595 nm) (Nexcelom Bioscience LLC, Lawrence, MA).

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 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: Following washing and centrifugation, the cell pellet was suspended in PBS and the intensity of fluorescence for each cell was quantified on a Cellometer Vision cell counter using filter cube VB-595–502 (excitation/emission: 525/595 nm) (Nexcelom Bioscience LLC, Lawrence, MA).

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