Figure 2.

Low concentrations of inactivated F. nucleatum promote HTR8/SVneo invasion; high concentration of inactivated F. nucleatum impairs migration of HTR8/SVneo cells. HTR8/SVneo cells were stimulated with F. nucleatum for 6 h using indicated bacteria:trophoblast ratios. After culture in methyl cellulose-containing medium, spheroids were embedded in matrigel and observed to analyse invasive behaviour (A, B). Bar graph shows relative sprouting expansion after 48 h normalized to spheroid size at 0 h (A). Data are presented as mean ± SEM and were analysed by Repeated Measures ANOVA with Dunnett’s multiple comparison post test, comparing each treatment against the corresponding control. *p_adj < 0.05 Representative microscopic images are shown (B). Experiments were performed 6 times. Scratch assay was performed to assess the migratory behaviour of bacteria-treated trophoblasts (C–E). EGF was used as positive control. Inactivated bacteria were added in different ratios (0.01; 0.1; 1; 10 bacteria per trophoblast cell). Bar graphs represent relative area recovered by HTR8/Svneo treated with either F. nucleatum (above) or E. coli (below) after 12 h (C) or BeWo treated with F. nucleatum after 30 h (E) normalized to unstimulated control. Data are presented as mean ± SEM. *p_adj < 0.05; ***p_adj < 0.001 as analysed by Repeated Measures ANOVA with Dunnett’s multiple comparison post test, comparing each treatment against the corresponding control. Experiment was performed 6 times in quadruplicate (C) or triplicate (E). Each point represents the mean value of the replicates for each experiment. Representative microphotographs of HTR8/SVneo taken with a 10 × objective taken after 0 and 12 h of the scratch (D). EGF, epidermal growth factor; Ctl, control.