Figure 2
Butyrate down-regulates NRP-1 at the mRNA and protein level. HCT116, HT29 and Caco-2 cell lines were treated for 20 hours with media supplemented with increasing concentrations of butyrate. (A) NRP-1 mRNA expression in all three cell lines is significantly reduced when treated with butyrate compared to untreated cells. (B) The level of NRP-1 protein was measured in samples from cells treated with the same range of butyrate concentrations (Bi) shows typical immunoblot for NRP-1 and actin loading control. Intensity was quantitated, normalized to actin and expressed as a proportion of the untreated control (Bii). Reduction was significant at 10 mM and above. *P < 0.05, **P < 0.01, ***P < 0.001. (C) HCT116 cells were grown in 96 well plates, fixed and stained for NRP-1 following butyrate treatment. Panels I & ii show sample images acquired during HCA of the untreated control sample with distinct distributions of NRP-1; (D) Amount of NRP-1 immunofluorescence in cells was quantified by HCA following treatment with butyrate. Significance of decrease across three experiments was determined by ANOVA with p-values inset in the figures.