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. 2021 Apr 6;12(2):e03491-20. doi: 10.1128/mBio.03491-20

FIG 5.

FIG 5

Removal of ED rescues E. coli inv invasion into MUC1-ΔCT cells. (A) Western blot analysis of DOX+ MUC1-ΔCT cells induced with 10 μg/ml doxycycline for 24 h, treated with 5 μg/ml StcE or E447D for 2 h and stained with α-MUC1-ED antibody 214D4 or actin. (B) Flow cytometry of DOX+ MUC1-ΔCT cells treated with 5 μg/ml StcE or E447D for 2 h and stained with α-MUC1-ED antibody 139H2. (C and D) Immunofluorescence confocal microscopy of DOX+ MUC1-ΔCT cells treated with StcE or E447D, stained with α-MUC1-ED antibody 139H2 (green) (C) or α-MUC1-SEA antibody 232A1 (green) (D) and DAPI to stain the nuclei (blue). White scale bars represent 20 μm. (E) Flow cytometry of representative DOX− MUC1-ΔCT cells and DOX+ MUC1-ΔCT cells treated with StcE or E447D, infected with E. coli inv (GFP) at MOI 20 for 2 h, and stained for MUC1 with α-MUC1-ED antibody 139H2. (F) Total infected cell percentage (Q2+Q3) in E447D-or StcE-treated DOX− MUC1-ΔCT cells and DOX+ MUC1-ΔCT cells calculated from (E). Each pair of data points represents the result from a single independent experiment. Three independent experimental replicates are shown. (G) Total infected cell percentage (Q2+Q3) in E447D- or StcE-treated DOX− MUC1-YF cells and DOX+ MUC1-YF cells. Each pair of data points represents the result from a single independent experiment. Four independent experimental replicates are shown. (H) Total infected cell percentage (Q2+Q3) in E447D- or StcE-treated DOX− MUC1-CT33 cells and DOX+ MUC1-CT33 cells. Each pair of data points represents the result from a single independent experiment. Four independent experimental replicates are shown. Statistical significance was determined by Student’s t test using GraphPad Prism software. *, P < 0.05; **, P < 0.01; ns, not significant.