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: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

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: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

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: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

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: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

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: Human MDR1 transfection HEK293 cells were transfected with the pCMV6-NEO plasmid containing no insert (empty vector, EV) or the full-length human MDR1 gene (Origene, Rockville, MD) using lipofectamine LTX and PLUS reagents (Invitrogen, Carlsbad, CA).

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