Journal: Nature Communications
Article Title: Circadian control of bile acid synthesis by a KLF15- Fgf15 axis
doi: 10.1038/ncomms8231
Figure Lengend Snippet: ( a ) In situ hybridization analysis of Klf15 and Fgf15 expression in Klf15 +/+ and Klf15 −/− mouse ileum. White arrows indicate Klf15 or Fgf15 mRNA signal. ( b ) Effects of adenoviral overexpression and shRNA knockdown of Klf15 on Fgf15 mRNA expression in primary mouse small intestinal epithelial cells. Primary small intestinal epithelial cells were infected with control virus ( Ctl ), ad Klf15 , or sh Klf15 as indicated. ( c ) Fgf15-Luc reporter assay in Caco2 cells following heterologous overexpression of Flag-Klf15 ( Klf15 ) or pc-DNA3.1 (control, Ctl ). ( d ) ChIP analysis of KLF15 ( Flag-Klf15 ) binding to designate regions of the Fgf15 and the Kv channel-interacting protein 2 ( KChip2 ) promoters in the mouse colon cell line CT26. The relative binding abundance (Rel abundance) was normalized to IgG. ( e ) Fgf15 mRNA expression in Klf15 +/+ versus Klf15 −/− mouse intestine segments: Duo, Jej, ileum and colon ( n =8). ( f ) Circadian rhythm of Klf15 mRNA expression in Klf15 +/+ mouse ileum ( P <0.01) was abolished in Klf15 −/− mouse ileum ( n =5 per time point). ( g ) Immunoblot of circadian KLF15 protein expression in the ileum (representative of three experiments). Ileal Fgf15 mRNA expression ( h ) and blood FGF15 protein concentrations (Conc) ( i ) exhibit circadian variations in Klf15 +/+ mice ( P <0.01) but the rhythms in Klf15 −/− mice were lost with increased expression at indicated time points ( n =5). Data represent mean±s.e.m. Statistical significance of circadian rhythm for each genotype was analysed using analysis of variance followed by Bonferroni post-test. Statistical significance between the two individual groups was assessed using Student's t- test. * indicates P <0.05, compared with control ( Ctl ), or Klf15 +/+ mice. Scale bars, 100 μm ( a ).
Article Snippet: Primary mouse small intestine epithelial cells were purchased from Cell Biologics Inc. (Chicago, IL, USA) and cultured in epithelial cell medium containing 0.1% epidermal growth factor, 0.1% insulin-transferrin sodium selenite, 0.1% hydrocortisone, 5% FBS, 1% L-glutamine and a 1% antibiotic-antimycotic solution on gelatin (Sigma-Aldrich, St Louis, MO, USA)-coated plates.
Techniques: In Situ Hybridization, Expressing, Over Expression, shRNA, Knockdown, Infection, Control, Virus, Reporter Assay, Binding Assay, Western Blot
