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Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in <t>OATP1B1-overexpressing</t> HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.
Estradiol [6,7 3h(N)] 17β D Glucuronide, supplied by American Radiolabeled Chemicals Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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3 h]-estradiol-17β-glucuronide  (American Radiolabeled Chemicals Inc)
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Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in <t>OATP1B1-overexpressing</t> HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.
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estradiol-17β-d-glucuronide (eβg)  (Millipore)
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Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in <t>OATP1B1-overexpressing</t> HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.
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Estradiol 17β D Glucuronide (E 2 17βg, Disodium Salt, supplied by GlpBio Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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estradiol-17β-glucuronide  (Toronto Research Chemicals)
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Toronto Research Chemicals estradiol-17β-glucuronide
Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in <t>OATP1B1-overexpressing</t> HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.
Estradiol 17β Glucuronide, supplied by Toronto Research Chemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in OATP1B1-overexpressing HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.

Journal: Cancer Research Communications

Article Title: Systematic Evaluation of Tyrosine Kinase Inhibitors as OATP1B1 Substrates Using a Competitive Counterflow Screen

doi: 10.1158/2767-9764.CRC-24-0332

Figure Lengend Snippet: Development and validation of the CCF method. A, Uptake of 0.01 μmol/L [ 3 H]-EβG in OATP1B1-overexpressing HEK293 cells was evaluated at room temperature over 120 minutes in 96-well plates ( n = 4 technical replicates, representative of n = 2 biological replicates; error bars represent SD). B, After saturation of cells at 1 hour, 100 μmol/L unlabeled EβG was spiked into wells, causing efflux of [ 3 H]-EβG ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SD). C, After 1 hour of saturation with 0.01 μmol/L [ 3 H]-EβG, 100 μmol/L positive (blue bars) and negative (gray bars) control substrates of OATP1B1 were spiked into wells and incubated for 30 minutes. Final intracellular radioactivity was measured and presented as relative to untreated control ( n = 9 technical replicates across n = 3 biological replicates; error bars represent SD. ***, P < 0.001; ****, P < 0.0001; five compared with the control). D, Stimulated efflux of preloaded [ 3 H]-EβG stimulated after the addition of 10 μmol/L TKI. Final intracellular radioactivity was measured and presented as relative to the efflux induced by an equimolar concentration of EβG, a known substrate and efflux inducer ( n = 6 technical replicates across n = 2 biological replicates; error bars represent SEM). VC, vector control.

Article Snippet: Transport function was assessed using a radiolabeled prototypical substrate for OATP1B1, estradiol [6,7- 3 H(N)]-17β-D-glucuronide (EβG; specific activity, 50 Ci/mmol; purity, 99%; American Radiolabeled Chemicals).

Techniques: Biomarker Discovery, Control, Incubation, Radioactivity, Concentration Assay, Plasmid Preparation

Using a biomarker and in silico docking to evaluate pazopanib as a substrate. Kinase activity of VEGFR kinase from OATP1B1-overexpressing and vector control (VC) cells ( A ) and hepatocytes isolated from WT and OATP1A/1B-KO mice (error bars represent SD; ***, P < 0.001; B ) following a 1-hour treatment with 10 μmol/L pazopanib. Kinase activity was assessed by ADP-Glo (error bars represent SD; ****, P < 0.0001). Molecular in silico docking of pazopanib in elucidated OATP1B1 structure ( C ) and with E3S superimposed ( D ).

Journal: Cancer Research Communications

Article Title: Systematic Evaluation of Tyrosine Kinase Inhibitors as OATP1B1 Substrates Using a Competitive Counterflow Screen

doi: 10.1158/2767-9764.CRC-24-0332

Figure Lengend Snippet: Using a biomarker and in silico docking to evaluate pazopanib as a substrate. Kinase activity of VEGFR kinase from OATP1B1-overexpressing and vector control (VC) cells ( A ) and hepatocytes isolated from WT and OATP1A/1B-KO mice (error bars represent SD; ***, P < 0.001; B ) following a 1-hour treatment with 10 μmol/L pazopanib. Kinase activity was assessed by ADP-Glo (error bars represent SD; ****, P < 0.0001). Molecular in silico docking of pazopanib in elucidated OATP1B1 structure ( C ) and with E3S superimposed ( D ).

Article Snippet: Transport function was assessed using a radiolabeled prototypical substrate for OATP1B1, estradiol [6,7- 3 H(N)]-17β-D-glucuronide (EβG; specific activity, 50 Ci/mmol; purity, 99%; American Radiolabeled Chemicals).

Techniques: Biomarker Discovery, In Silico, Activity Assay, Plasmid Preparation, Control, Isolation