I., Tanguay R. exhibited AhR antagonist activity for induction of CYP1A1. Simulations suggest that while quercetin and apigenin interact primarily with the same residues, the strength of interactions between specific AhR residues with CYP1A1 agonist, quercetin, in comparison with CYP1A1 antagonist, apigenin, is different; thus, such interactions are presumably indicative of potential switches for modulating CYP1A1 activity. The structure-dependent effects of the hydroxyl flavonoids on induction of UGT1A1 were similar to that observed for induction of CYP1A1 except that luteolin and apigenin induced UGT1A1 levels similar to that observed for TCDD, whereas both compounds were AhR antagonists for CYP1A1. Thus, the effects of the flavonoids in Caco2 cells on Ah-responsiveness and interactions with butyrate were both ligand structure- and response-dependent and these activities are consistent with hydroxyflavonoids being selective AhR modulators. induction), gossypetin (10?M; higher concentrations were toxic), and luteolin, kaempferol, and apigenin (10?M; higher concentrations were poorly soluble). Chromatin immunoprecipitation assay The chromatin immunoprecipitation (ChIP) assay was performed using ChIP-IT Express Magnetic Chromatin Immunoprecipitation kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Caco2 cells were treated with sodium butyrate overnight, and TCDD was subsequently added into the media for 2? h prior to cell harvest. Cells were then fixed with 1% formaldehyde, and the cross-linking reaction was stopped by addition of 0.125?M glycine. After washing twice with phosphate-buffered saline, cells were scraped and pelleted. Collected cells were hypotonically lysed, and nuclei were collected. Nuclei were then sonicated to desired chromatin length (200C1500?bp). The sonicated chromatin (25?g) was immunoprecipitated with primary antibodies (25?g) and protein A-conjugated magnetic beads at 4C for 12?h. After the magnetic beads were extensively washed, protein-DNA crosslinks were reversed and eluted. DNA was prepared by proteinase K digestion followed by PCR amplification. The human primers were 5-TCA ATC AAG AGG CGC GAA CCT C-3 (sense), and 5-CTA CAG CCT ACC AGG ACT CG-3 (antisense), and then amplified by targeting a 203-bp region of human promoter which contained the AhR binding sequences. The human primers were 5-GTG TTA TCT CAC CAG AAC AAA-3 (sense) and 5-TAC CCT CTA GCC ATT CTG-3 (antisense), and subsequently amplified by targeting a 190-bp region of human promoter, which contained the AhR-binding sequences. PCR products were resolved on a 2% agarose gel in the presence of ETBR. Quantitative real-time reverse transcriptase PCR cDNA was prepared from the AR-9281 total RNA of cells using High Capacity RNA-to-cDNA Kit (Applied Biosystems, Foster City, CA). Each PCR was carried out in triplicate using Bio-Rad SYBR Universal premix for 1?min at 95C for initial denaturing, followed by Itga2 40 cycles of 95C for 15?s and 60C for 1?min in the Bio-Rad iCycler (MyiQ?2) real-time PCR System. The comparative CT method was used for relative quantitation of samples. Values for each gene were normalized to expression levels of TATA-binding protein (TBP). The sequences of the primers used for real-time PCR were as follows: sense 5-GAC CAC AAC CAC CAA GAA C-3, antisense 5-AGC GAA GAA TAG GGA TGA AG-3; sense 5-GAA TCA ACT GCC TTC ACC AAA AT-3, antisense 5-AGA GAA AAC CAC AAT TCC ATG TTC T-3; sense 5-GAT CAG AAC AAC AGC CTG CC-3, antisense 5-TTC TGA ATA GGC TGT GGG GT-3. Western blot analysis Cells (3105) were plated in 6-well plates in DMEM media made up of 2.5% FBS for 24?h and then treated with different concentrations of the compounds. Cellular lysates were prepared in lysis buffer made up of 50?mM HEPES, 0.5?M NaCl, 1.5?mM MgCl2, 1?mM EGTA, 10% glycerol, and 1% Triton-X-100, each 1 protease and phosphatase inhibitor cocktail (GenDEPOT), and 1% NP-40. The cells were disrupted and extracted at 4C for 30?min. After centrifugation, the supernatant was obtained as the cell lysate. Protein concentrations were measured using AR-9281 the Bio-Rad protein assay. Aliquots of cellular proteins were electrophoresed on 10% SDSCpolyacrylamide gel electrophoresis (PAGE) AR-9281 and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membrane was allowed to react with a specific antibody, and detection of specific proteins was carried out by enhanced chemiluminescence. Loading differences were normalized using a polyclonal -actin or GAPDH antibody. Generation of AhR-deficient Caco2 cells Two AhR CRISPR guideline RNAs, in a Cas9 vector (pSpCas9 BB-2A-GFP PX458) which also expresses GFP, were purchased from GenScript (Piscataway, NJ). Sequences of the guideline RNAs were 5-AAG TCG GTC TCT ATG CCG CT-3 and 5-AGA CCG ACT TAA TAC AGA GT-3. Caco2 cells were transfected with AR-9281 each plasmid and 48?h later, cells were sorted by flow cytometry to collect the 5% highest GFP expressing cells. Clonal cells were grown and.