Figure 1.
After co-culture of Caco-2 cells with L casei and B. breve and hybridization on Human U133A genechip, results were normalized with RAM and analysis was performed using dChip software as described in material and method section. Significantly modulated genes (fold change >1,75 and p-value <0,05) are shown. Numbers indicate the modulated genes. A: global gene expression modulation; B: cell cycle gene expression modulation; C: Hierarchical clustering of cell cycle gene expression was performed using dChip software with Euclidian distance and average as a linkage method. Before clustering, expression values for one gene across all samples were standardized to produce a mean of zero. Increased or decreased values were then compared with that mean. Red and blue colors represent expression that is higher or lower than the mean value, respectively. The key for intensity of expression is indicated under the bar. See Table S2 for the corresponding fold change and p-value of cell cycle modulated genes.
Figure 2.
Fold change of gene expression after overnight co-culture of m-ICcl2 with L.casei and B.breve.
qRT-PCR was analyzed by the ddCt method using m-ICcl2 alone and GAPDH as reference.
Figure 3.
Identification of effectors that affect m-ICcl2 cell cycle related gene.
qRT-PCR was analyzed by the ddCt method using m-ICcl2 alone and GAPDH as reference.
Figure 4.
Impact of acetate and lactate on m-ICcl2 cyclin D1 and cyclin E1 gene expression.
qRT-PCR was analyzed by the dδCt method using m-ICcl2 alone and GAPDH as reference.
Figure 5.
Acetate induces cell proliferation arrest in a concentration & pH dependent manner.
A: Number of m-ICcl2 per well after incubation at different pH with 20 mM of SCFA. Number represent cell counts (x105) per well. B-C: qRT-PCR of cyclin D1 (B) and cyclin E1 (C) gene expression after incubation of m-ICcl2 with or without 20 mM acetate or lactate at different pHs. qRT-PCR was analyzed by the dδCt method using m-ICcl2 alone and GAPDH as reference.
Figure 6.
Acetate and pH induces respectively a down-regulation of cyclin D1 and cyclin E1 proteins.
Western blots were performed after 16 hr co-culture of m-ICcl2 cells with 20 mM of acetate and lactate at different pHs. For the detection of cyclin E1, cells were synchronized by a double thymidine block treatment prior co-culture as described in material and methods section.