Butyrate down-regulates VEGF at the mRNA and protein level. Human colon cancer cell lines were treated with increasing concentrations of butyrate. RNA and protein samples for analysis were described as in fig 2. (A) Butyrate significantly reduced VEGF mRNA expression compared to untreated cells in HCT116 and Caco-2 cell lines whereas there was no significant difference of VEGF expression in HT29 cells. (B) Supernatants of HCT116 cultures treated with increasing concentrations of butyrate were assessed for VEGF levels by ELISA. (Bi) shows the absolute quantitation of VEGF, (Bii) shows the VEGF level normalised to cell count. (C) The level of VEGF protein was established in samples from cells treated with the same range of butyrate concentrations (Ci), shows sample immunoblots of VEGF and actin loading control. Intensity was quantitated, normalized to actin and expressed as a proportion of the untreated control (Cii). *P < 0.05, **P < 0.01. (D) Levels of VEGF in cells and at subcellular locations were determined by HCA. The means ± SD of three independent repeat experiments are shown for total VEGF (subpanel i); perinuclear VEGF (subpanel ii) and cytoplasmic VEGF (subpanel iii). Significance of decrease across three experiments was determined by ANOVA with p-values inset in the figures. Sample HCA images showing distribution of VEGF in untreated cells (subpanel iv) and following treatment with 10 mM butyrate (subpanel v). Butyrate treatment resulted in a more punctuate staining pattern (see arrows).