Bulk RNAseq analysis of butyrate and/or secondary bile acid treatment of T84 cell line

P-glycoprotein is a key component of the intestinal epithelium playing a pivotal role not only in the removal of toxins but also in the efflux of endocannabinoids to prevent excessive inflammation and sustain a homeostatic tone within the intestine. Previous studies have shown short-chain fatty acids and secondary bile acids produced by the intestinal microbiota potentiate induction of P-glycoprotein expression, yet the mechanism remains to be elucidated. Here, we employ RNA sequencing to evaluate the transcriptome signature triggered by each metabolite alone, and unique pathways activated by the metabolite combination. We show butyrate regulates NRF2 signaling and pathways related to chromatin modifications, while bile acids activate nuclear receptor pathways including vitamin D receptor and pregnane X receptor. In combination these metabolites activate a unique transcriptional program involving multiple pathways including transcription factors and kinases that converge on P-glycoprotein induction. T84 cells were seeded from six separate flasks for six biological replicates. Cells were treated with 5mM butyrate, 50µM each of three secondary bile acids together (lithochoilc acid, deoxycholic acid, ursodeoxycholic acid) or a combination of all four metabolites at the listed concentrations. DMSO vehicle group served as a control. 24 samples of 4 conditions with 6 biological replicates each.