Fig 1.
Lactobacillus growth varies by media type.
Lactobacillus strains were grown overnight, washed in PBS, and inoculated into fresh: A) MRS media; B) TSA + 1% Dextrose; or C) HESC 2/0 media. The optical density 595 (OD595) was measured every 30 minutes for 48 hours using a spectrophotometer. For each graph, the Area Under the Curve (AUC) was calculated using GraphPad Prism 6. Differences were determined by comparing the AUC of three biological replicates by unpaired ANOVA (*p<0.05). Error bars represent the standard deviation between three independent biological experiments.
Fig 2.
Lactobacillus species differ in the ability to form biofilms depending on media type.
Lactobacillus was grown overnight, washed in PBS and incubated in: A) TSB with 5% dextrose; or B) MRS media for 48 hours in a 96-well plate. Normalized absorbance was calculated by subtracting the OD595 from the control well and multiplying by four to account for dilutions. Experiments were performed in biological triplicates of technical quadruplicates. Error bars represent standard deviation between biological trials. Significance was determined by an unpaired ANOVA (p<0.05) after confirmation of a normal distribution of the data via the Shapiro-Wilk test.
Fig 3.
Lactobacillus associates with decidualized human endometrial stromal cells (dT-HESCs).
A) The dT-HESCs were incubated with Lactobacillus at a MOI of 10 for two hours. The percent of associated bacteria was calculated relative to the total number of bacteria in the well. B) Lactobacillus were incubated for two hours in the presence of Triton-X and colony forming units (CFUs/ml) were quantified. Experiments were completed in biological quadruplets of technical triplicates. Error bars represent standard deviation between biological trials. Error bars represent standard deviation between biological trials, and significance differences were tested using an unpaired ANOVA (*p<0.05).
Fig 4.
Lactobacillus affects total p38 and phosphorylated (Phospho) levels of p38.
dT-HESCs were incubated with Lactobacillus at a MOI of 10 for three hours. Protein lysates were collected and analyzed via Western Blotting using densitometry in Image J to determine the amounts of A) total p38 and B) total Phospho p38. A β-tubulin internal loading control was used to account for loading differences by normalizing levels per well to the total protein levels. Graphed densitometry data is presented as a percent of the mock control for each of three biological replicates. Error bars represent the standard deviation of the data. Significance was determined using an unpaired ANOVA and a post-hoc Dunnett’s test.
Fig 5.
Lactobacillus does not induce dT-HESC death.
dT-HESCs were incubated with Lactobacillus at a MOI of 10 and incubated for four hours. Cell permeability was detected using an ethidium homodimer assay, and percent permeability was calculated by lysing the remaining cell in each well. Group B streptococcal strain, GB00411, was included as a positive control. Graphed data represents three biological replicates, and the error bars represent the standard deviation of the data. Significance was determined using an unpaired ANOVA.