Streptococcus gallolyticus subsp. gallolyticus promotes colorectal tumor development
Fig 5
Sg promotes cell proliferation in a β-catenin dependent manner.
A. Knockdown of β-catenin abolished the effect of Sg. Cell proliferation assays were performed as described in the legend for Fig 1. Untransfected HT29 cells, β-catenin stable knockdown HT29 cells (HT29-B1) or HT29 cells transfected with a control shRNA (HT29-C1) were used. Data was analyzed using two-way two-tailed ANOVA followed by SNK test. Significance shown panel A is for comparison between HT29 cells co-cultured with TX20005 and HT29-B1 with TX20005. B—D. Inhibition of β-catenin transcriptional activity by iCRT3 rendered cells unresponsive to Sg. B. Stationary phase TX20005 or L. lactis bacteria were added to the wells (~1x102 cfu/well) in the presence or absence of iCRT3 (25 μM), incubated for 24 hours and viable cells enumerated. C and D. Stationary phase TX20005 or L. lactis were added to cells (~105 cfu/well) as described in the legend for Fig 4 and incubated for 12 hours in the presence or absence of iCRT3. Total cell lysates were prepared and subject to western blot assays to compare β-catenin, c-Myc and PCNA protein levels. Representative images are shown (C). Band intensity was quantified using Image J, normalized to β-actin first and then to the cells only control (D). Data are presented as the mean ± SEM. Each experiment was done with duplicate wells and was repeated at least three times. Data in panels B and D were analyzed by using unpaired, two-tailed t test. *, p < 0.05; **, p < 0.01.