Fig 4. MUC1 and MUC16 are expressed on the surface of human airway organoid-derived air-liquid interface cultures and decreases upon StcE treatment.

(A) Microscopy of permeabilized human airway organoid-derived air-liquid interface cultures showing combined extracellular and intracellular staining of MUC1, MUC4, MUC5AC and MUC16. (B) Microscopy of live stained air-liquid culture for MUC1, MUC4, MUC5AC and MUC16 without permeabilization. MUC1 and MUC16 are detectable demonstrating expression on the cell surface, whereas MUC4 staining is negative and MUC5AC only stains positive in occasional mucus strands on top of the cells. (C) Immunoblot analysis of MUC1 levels in human airway organoid-derived ALI cultures from donor 1 and donor 2 treated with StcE, E447D or no treatment. The high molecular weight MUC1 is removed upon StcE treatment. (D) Microscopy of MUC1 and MUC16 in permeabilized ALI cultures along with O-glycan probe X409-GFP. Arrows indicate co-localization of X409 with MUC16, but not MUC1. (E) Quantification of colocalization of MUC1 and MUC16 with X409 staining in ALI cultures of two donors as performed in D. Mander’s overlap coefficient plots are included in S3 Fig. (F) Microscopy of surface binding of X409 on untreated, 10ug/ml StcE and 10ug/ml E447D treated ALI cultures. All white scale bars indicate 50 μm. (G) Quantification of X409 signal intensity per imaged field from experiment performed in E. Statistical analysis was performed by repeated measures one way-ANOVA with Tukey’s post-hoc test. p<0.05 [*].