Balkan Journal of Medical Genetics

IN VITRO ANALYSIS OF AKR1D1 INTERACTIONS WITH CLOPIDOGREL: EFFECTS ON ENZYME ACTIVITY AND GENE EXPRESSION
Shutevska K1*, Kadifkova Panovska T1, Zhivikj Z1, Kapedanovska Nestorovska A2
*Corresponding Author: *Corresponding Author: Kristina Shutevska, Majka Tereza 47, 1000 Skopje, Republic of North Macedonia, +389 2 1326032 (142), k.sutevska@ff.ukim.edu.mk
page: 69

MATERIALS AND METHODS

Reagents and Chemicals – Molecular Cloning Reagents: l Full-length AKR1D1 cDNA (OriGene, Rockville, MD, USA; Cat# RC223056): Used for amplify- ing and cloning the AKR1D1 gene into an expres- sion vector. l pET28b+ vector (Novagen (Merck KGaA), Darmstadt, Germany; Cat# 69865-3): Expression vector used for recombinant protein production. – Enzyme Assay Reagents: l Testosterone (Sigma-Aldrich, St. Louis, MO, USA; Cat# T1500): Substrate for AKR1D1 ac- tivity assays. l Clopidogrel Bisulfate (Sigma-Aldrich, St. Louis, MO, USA; Cat# C0612): Evaluated for interac- tions with AKR1D1 at both enzymatic and tran- scriptional levels. l 2-oxoclopidogrel (Cayman Chemical, Ann Ar- bor, MI, USA; Cat# 20394): Metabolite of clopi- dogrel tested for interactions with AKR1D1 at both enzymatic and transcriptional levels. – Cell Culture Reagents: l HepG2 Hepatocyte Cell Line (ATCC, Manassas, VA, USA; Cat# HB-8065): An in vitro model system used to study AKR1D1 expression and hepatic metabolism. l Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich, St. Louis, MO, USA; Cat# D6429): Basal medium for HepG2 cell mainte- nance. l Fetal Bovine Serum (FBS) (Capricorn Scientific, Ebsdorfergrund, Germany; Cat# FBS-12A): Sup- plemented in DMEM to support cell growth and viability. l Trypsin-EDTA Solution (Lonza, Walkersville, MD, USA; Cat# CC-5012): Used for cell detach- ment during subculturing. – MTT Assay Reagents: l MTT Solution (5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA; Cat# M2128): Used to evaluate cell viability through formazan crystal formation. l Dimethyl Sulfoxide (DMSO) (Thermo Fisher Scientific, Waltham, MA, USA; Cat# D12345): Used to dissolve formazan crystals for spectro- photometric analysis. l NADPH (Sigma-Aldrich, St. Louis, MO, USA; Cat# N7505): Essential cofactor for spectrofluo- rometric enzyme activity assays. –Expression Assay Reagents l HpaI Restriction Enzyme (New England Biolabs, Ipswich, MA, USA; Cat# R0105S): Used for linearizing the AKR1D1 plasmid to generate a calibration curve for qRT-PCR quantification. l Shrimp Alkaline Phosphatase (Takara Bio, Shiga, Japan; Cat# 2650A): Used for dephosphorylat- ing the linearized plasmid DNA to prevent self- ligation. l TRI Reagent® (Sigma-Aldrich, St. Louis, MO, USA; Cat# T9424): Used for total RNA extrac- tion from HepG2 cells. l ProtoScript® II Reverse Transcriptase (New Eng- land Biolabs, Ipswich, MA, USA; Cat# M0368): Used to synthesize complementary DNA (cDNA) from isolated RNA. l XCEED qPCR SG 2x Mix Lo-ROX (Institute of Applied Biotechnologies, Prague, Czech Repub- lic; Cat# QR0100): Used for quantitative real- time PCR analysis of AKR1D1 gene expression. l Forward and Reverse Primers (GeneriBiotech, Hradec Králové, Czech Republic): Specific prim- ers designed for the amplification of AKR1D1 during quantitative real-time PCR.

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