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. 2020 Jul 21;20(14):4050. doi: 10.3390/s20144050

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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IC₅₀ of Furafylline for the inhibition of CYP1A2 enzyme expressed within yRD⁺ and yRD⁻:: Bax cells. Furafylline’s IC₅₀ for inhibition of CYP1A2 enzyme was expressed within whole cells. The cells that were used were from strains (A) yRD⁺ (+CPR) and (B) yRD⁻:: Bax (–CPR) were transformed with an episomal plasmid encoding a CYP1A2 gene expression cassette, CYP1A2 enzyme expression was driven by the ethanol-inducible ADH2 promoter (ADH2p). In yRD⁻ cells, Bax expression was driven by the GAL1 promoter (GAL1p), which was de-repressed in ethanol. Recombinant cells were grown for 18 h in YPD liquid medium containing glucose, allowing for around 6 h of induction and de-repression of ADH2p and GAL1p in ethanol (it takes ~12 h for glucose to be utilized and converted completely to ethanol). Cells were harvested and washed in PBS-saline. The assay included 1 × 10⁷ washed cells and 3-cyano-7-ethoxycoumarin (CEC) as a substrate. Each point represents the mean of triplicate readings; bars denote ± standard deviation. The x-axis represents the values of the concentration of furafylline in µM (ranges from 0.1 to 100 µM) that were incubated with cells for 10 min, the y-axis relative fluorescence units of the fluorescent product formed. The IC₅₀ values were calculated using the GraphPad Prism software. Furafylline’s IC₅₀ for inhibition of CYP1A2 Supersomes (insect cell-derived CYP1A2-bearing microsomes; Corning-Gentest), with a 10 min incubation with a microsomal enzyme, is 6.1 µM [36,37]. (C) Western Blotting of lysates from yRD⁺, yRD⁻, and yRD⁻:: Bax cells expressing CYP1A2. Yeast cell lysates (5 µg of total cellular protein for monitoring CYP1A2 and actin; 50 µg for Bax) fractionated on SDS/PAGE and probed with antibodies to CYP, Bax, and actin proteins after Western blotting. (aCell lysates probed with a CYP1A2 antibody (Proteintech, 19936-1-AP). Lane 1, BC600:: GAL1p-Bax (lysate from untransformed cells, which did not contain CYP1A2 episomal plasmid, as negative control). Lanes 2-4: cell lysates from the strains BC300, BC600, and BC600:: GAL1p-Bax transformed with an episomal plasmid that contained a CYP1A2 gene expression cassette under the control of the ADH2 promoter. Lane 2, BC300:: CYP1A2, lane 3, BC600:: CYP1A2, lane 4, BC600:: Bax,CYP1A2, and lane 5, 1.5 pmole of microsomal CYP1A2 (as positive control; CYP Design Ltd.). (b) Lanes 2-4, as in (a), probed cell lysates with an actin antibody (Proteintech, 60008-1-Ig). (c) Lanes 2-4, same as in (a), the cell lysates being probed with a c-myc antibody tagged to the human Bax protein (Thermo Scientific, MA 1-980).

IC₅₀ of Furafylline for the inhibition of CYP1A2 enzyme expressed within yRD⁺ and yRD⁻:: Bax cells. Furafylline’s IC₅₀ for inhibition of CYP1A2 enzyme was expressed within whole cells. The cells that were used were from strains (A) yRD⁺ (+CPR) and (B) yRD⁻:: Bax (–CPR) were transformed with an episomal plasmid encoding a CYP1A2 gene expression cassette, CYP1A2 enzyme expression was driven by the ethanol-inducible ADH2 promoter (ADH2p). In yRD⁻ cells, Bax expression was driven by the GAL1 promoter (GAL1p), which was de-repressed in ethanol. Recombinant cells were grown for 18 h in YPD liquid medium containing glucose, allowing for around 6 h of induction and de-repression of ADH2p and GAL1p in ethanol (it takes ~12 h for glucose to be utilized and converted completely to ethanol). Cells were harvested and washed in PBS-saline. The assay included 1 × 10⁷ washed cells and 3-cyano-7-ethoxycoumarin (CEC) as a substrate. Each point represents the mean of triplicate readings; bars denote ± standard deviation. The x-axis represents the values of the concentration of furafylline in µM (ranges from 0.1 to 100 µM) that were incubated with cells for 10 min, the y-axis relative fluorescence units of the fluorescent product formed. The IC₅₀ values were calculated using the GraphPad Prism software. Furafylline’s IC₅₀ for inhibition of CYP1A2 Supersomes (insect cell-derived CYP1A2-bearing microsomes; Corning-Gentest), with a 10 min incubation with a microsomal enzyme, is 6.1 µM [36,37]. (C) Western Blotting of lysates from yRD⁺, yRD⁻, and yRD⁻:: Bax cells expressing CYP1A2. Yeast cell lysates (5 µg of total cellular protein for monitoring CYP1A2 and actin; 50 µg for Bax) fractionated on SDS/PAGE and probed with antibodies to CYP, Bax, and actin proteins after Western blotting. (aCell lysates probed with a CYP1A2 antibody (Proteintech, 19936-1-AP). Lane 1, BC600:: GAL1p-Bax (lysate from untransformed cells, which did not contain CYP1A2 episomal plasmid, as negative control). Lanes 2-4: cell lysates from the strains BC300, BC600, and BC600:: GAL1p-Bax transformed with an episomal plasmid that contained a CYP1A2 gene expression cassette under the control of the ADH2 promoter. Lane 2, BC300:: CYP1A2, lane 3, BC600:: CYP1A2, lane 4, BC600:: Bax,CYP1A2, and lane 5, 1.5 pmole of microsomal CYP1A2 (as positive control; CYP Design Ltd.). (b) Lanes 2-4, as in (a), probed cell lysates with an actin antibody (Proteintech, 60008-1-Ig). (c) Lanes 2-4, same as in (a), the cell lysates being probed with a c-myc antibody tagged to the human Bax protein (Thermo Scientific, MA 1-980).