Figure 5.

HMGCS2 contributes to intestinal differentiation. (a) Caco-2 cells were transfected with empty vector (control) or transfected with Flag-HMGCS2 constructs. After 48 h, cells were lysed and extracted for protein. p21^Waf1, CDX2, H3K9ac, HMGCS2, Flag-tagged HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21^Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus control vector). (b and c) LS174T cells were infected with a recombinant adenovirus encoding the human HMGCS2 or vector control encoding GFP. After 48 h, cells were lysed and extracted for RNA and protein. (b) p21^Waf1, CDX2, H3K9ac, HMGCS2 and β-actin were determined by western blotting. The images are representative of three independent experiments. p21^Waf1, CDX2 and H3K9ac signals from three separate experiments were quantitated densitometrically and expressed as fold change with respect to β-actin. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). (c) CDX2 mRNA expression was assessed by real-time RT-PCR. (n=3, data represent mean±S.D.; *P<0.01 versus GFP control). Data are from one of three independent experiments with similar results. Overexpression of HMGCS2 inhibits HDAC and increases p21^Waf1 and CDX2 expression in Caco-2 and LS174T cells. (d) Immunohistochemical analysis of HMGCS2 protein expression in normal human small intestine. Human normal small intestine sections were fixed and stained with primary anti-human HMGCS2 antibody. HMGCS2 is specifically expressed in the more differentiated region (i.e., villus; arrows). Scale bars=50 μm. The images are representative of five cases