same time period. By contrast, 20567609 cells that were colonized with L. crispatus shifted their modes of cell division frequency between 7 and 11 hours; at those times, these cells exhibited an average of 2.3 cell divisions every 10 minutes with peaks of up to 5 cell divisions every 10 minutes. However, the increased number of cell divisions that was induced by L. crispatus did not alter the overall average proliferation rate. These data demonstrate the ability of L. crispatus to transiently enhance the frequency of cell division. S phase Progression is Reduced by L. rhamnosus and L. reuteri but not L. crispatus To further verify the finding that Lactobacillus colonization reduces cell cycle progression, the number of cells in S phase at the end of 24 hours of incubation was measured. Lactobacilli were allowed 9570468 to adhere to ME-180 cells for 24 hours. Bromodeoxyuridine was added to cells two hours prior to the termination of this assay, allowing the BrdU to be incorporated into the replicating DNA of cells in S phase. The adherence by all of the lactobacillus strains that were tested except for L. crispatus resulted in a reduced number of ME-180 cells in S phase. L. rhamnosus and L. reuteri reduced the level of BrdU incorporation to an average of 31% and 59%, respectively, relative to the BrdU incorporation level of control ME-180 cells. Thus, this finding once again demonstrates that colonization with L. rhamnosus and L. reuteri but not L. crispatus inhibits cell cycle progression in ME-180 cells. Lactobacilli Delay ME-180 Cells in G1 Phase To determine whether the bacterial-induced cell cycle delay occurred during a specific cell cycle phase, we analyzed cell phase profiles by flow cytometry. Bacteria were added to ME-180 cells for 24 hours, and cellular DNA was stained with propidium iodide. Four independent experiments were performed, and the percentages of cells in G1, S, and G2 were quantified. Representative flow cytometry histograms of the control ME-180 cells and the ME-180 cells that were colonized with the three Lactobacillus strains are presented in figure 4A. For the control ME-180 cells, an average of 57% of the examined cells were in G1 phase. Adhesion by L. rhamnosus and L. reuteri significantly increased the number of cells in G1 phase to an average of 62% and 63%, respectively. L. Lactobacilli Influence the Human Cell Cycle 7 Lactobacilli Influence the Human Cell Cycle crispatus colonization did not significantly alter the number of cells in G1 phase. L. rhamnosus Colonization Up-regulates p21 The G1 accumulation of cells is often mediated by increases in the activity of cyclin-dependent kinase inhibitors, such as p21. To investigate the role of p21 in lactobacillus-mediated cell cycle deceleration, lactobacilli were allowed to adhere to ME-180 cells for 24 hours. Bacteria-colonized cells were washed and prepared for either qPCR assay or immunofluorescence staining. Through the use of p21-specific primers for qPCR and the normalization of the results against the mRNA levels of a-tubulin or GAPDH, we demonstrate that L. rhamnosus upregulated p21 mRNA 2.5-fold in comparison to control cells. No significant change in p21 mRNA levels was observed in either L. reuteri or L. crispatus. The p21 protein is a cell cycle inhibitor, and its nuclear (S)-(-)-Blebbistatin site localization is associated with a slowing of the cell cycle. Indeed, 
compared with the control ME-180 cells, L. rhamnosus-colonized cells displayed a greater extent of p21 expression in their c