Fig. 1
Probiotic Bifidobacterium bifidum strains uniquely modify the MUC13 extracellular domain. (A) Immunoblot analysis of MUC13 modification/degradation and claudin-3 expression in HRT18 monolayers after incubation with different probiotic strains at MOI 10 for 20 h. The full-length 130 kDa MUC13- band that is used for quantification in (B) is marked by an arrow. (B,C) Quantification of protein expression of the 130 kDa MUC13 and 22 kDa claudin-3 bands (relative to uninfected control) in three biological replicates as depicted in A. All immunoblots used for quantification are available in Figure S1 and Figure S2. Statistical analysis was performed on non-normalized data using a one-way ANOVA with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. (D) Fluorescence confocal microscopy images of HRT18 cells stained in green with MUC13-ED (above) and MUC13-CT (below) antibodies after 20 h incubation with B. bifidum strains at MOI 10 and StcE. The maximal intensity projection is depicted. White scale bars represent 20 µM. (E) Quantification of GFP signal in cells stained with MUC13-ED and MUC13-CT antibodies as in (C). Statistical analysis was performed on non-normalized data using a one-way ANOVA with Tukey’s post hoc test. * p < 0.05; ** p < 0.01. (F) Immunoblot analysis of degradation of high-MW MUC13 (130 kDa) after 2 and 20 h incubation with B. bifidum W23, W28, and 20456 strains at MOI 10 and 50 in HRT18 cells. The full-length 130 kDa MUC13- band that is used for quantification in (G) is marked by an arrow. (G) Quantification of protein expression of MUC13 (relative to uninfected control) in three biological replicates as depicted in F. All immunoblots used for analysis are available in Figure S3. Statistical analysis was performed on non-normalized data using a one-way ANOVA or Krustal-Wallis test (when the data was not normally distributed) with Dunnett’s post hoc test to compare each bacterial sample to the uninfected control. * p < 0.05. Bacteria are listed in Table 1. All graphs depict the SEM of three independent experiments.
