[PMC free article] [PubMed] [CrossRef] [Google Scholar] 46. for the duration of the study. At of the study, WT and = 8, and = 8), RNA from proximal colonic mucosal scrapings was isolated using the RNAqueous-4-PCR kit (Ambion; AM1914), as per manufacturers instructions. Removal of DNA (Ambion; AM1906), Pifithrin-β quantification by Nanodrop spectrophotometry, and assessment of RNA integrity on a Bioanalyzer 2100 (Agilent Technologies) were performed. Samples (= 4, and WT, = 4) were randomized, Pifithrin-β and External RNA Control Consortium (ERCC) was added before beginning RNA sequencing library preparation. Sequencing libraries were generated using 250 ng of RNA and the TruSeq RNA Sample Preparation kit (Illumina) following manufacturers instructions. External RNA control consortium spike-in RNA control mix (Life Technologies) was added to the starting RNA. DNA libraries were pooled and sequenced on an Illumina HiSeq 2500 at the Texas AgriLife Genomics and Bioinformatics Services Facility (College Station, TX). Sequencing data were demultiplexed and aligned using Spliced Transcripts Alignment to a Reference software with default parameters and referenced against the genome of (Ensembl version GRCm38). Using EdgeR based on the matrix of gene counts resulted in a list of differentially expressed genes (DEGs) expressed as a ratio of value) below 0.1, and these DEGs were further analyzed in IPA based on their biological functions. Statistical Analysis All statistical analyses and figures were generated in GraphPad Prism version 8.1.2 for macOS, Pifithrin-β Graph Pad Software (La Jolla, CA; https://www.graphpad.com/). Means were compared using parametric or nonparametric methods according to compliance of normality (Shapiro-Wilk test). Outliers based on robust regression and outlier removal (ROUT) (test for comparing two means or one-way ANOVA followed by Tukeys multiple comparisons test for comparing three or more Rabbit polyclonal to PLEKHG3 means. Two-way ANOVA was used to test interaction effects between variables. Nonparametric methods include the Mann-Whitney U test for comparing two means or the Kruskal-Wallis test followed by Dunns multiple comparisons test for comparing three or more means. One-tailed values are reported in all analyses because of the directionality of our hypothesis. All values listed are group means, and error bars are presented as SE. Differences among groups were considered statistically significant when 0.05. Within figures, * 0.05, ** 0.01, *** 0.001, and the absence of * indicates 0.05. RESULTS Carcinogen-Induced DNA Damage is Not Associated With AhR Activity in Intestinal Epithelial Cells Since carcinogen activation is partially mediated by IECs (36), we investigated the effect of the loss of AhR in IECs immediately (12C72 h) following carcinogen exposure (Fig. 1= 0.2158). Representative images of DNA-damaged cells are shown in Fig. 1= 4 per each of the genotypes and time points. = Pifithrin-β 0.2158 (one-way ANOVA). and = 0.0088), as shown in Fig. 2= 0.0005) independently of the genotype (Fig. 2= 0.004) compared with the other treatment groups. Though the genotype (= 0.0102) and diet (= 0.0004) significantly altered the number of ACFs independently, collectively they did not have a significant interactive effect on ACF number (statistical interaction, = 0.4502; Fig. 2= 0.0198; Fig. 2= 0.0088 [Mann-Whitney (MW)]. = 0.0005 (MW). = 0.0004 (Kruskal-Wallis). No interaction between diet and genotype (= 0.4502), diet effect (= 0.0004), genotype effect (= 0.0102). = 0.0198 (MW). AhR, aryl hydrocarbon receptor; AOM, azoxymethane. Values are means??SE. * 0.05, ** 0.01, *** 0.001, and absence of * indicates 0.05. Descriptive statistics provided in Supplemental Table S3 (https://doi.org/10.6084/m9.figshare.11349614). Loss of AhR in IECs Induces Cell Proliferation in Colonic Crypts Pifithrin-β in the Premalignant Lesion Cohort Representative images of EdU+ proliferative cells at 15 wk after the last injection of AOM are shown in Fig. 3 0.0001) in the 0.0001). Furthermore, we found that both the genotype and the diet interactively modified cell proliferation (statistical interaction, = 0.0017; Fig. 3 0.01) but not at the top of the crypt (Fig. 3= 0.03) (Supplemental Fig. S4values, are reported in Supplemental Table S3 (https://doi.org/10.6084/m9.figshare.11349614). Open.