Extended Data Fig. 3: Validation of viral entry factor expression patterning across oral niches.

a, Immunofluorescence (IF) confocal microscopy demonstrated AQP5 (red, acini) colocalization with ACE2 (green) reveal that ACE2 is expressed on the apical (luminal) and basolateral membranes and is concentrated in the salivary gland (SG) acini/ducts (dotted/solid line, respectively; 1 independent replication). b-d, (b) Using healthy volunteer SG sections, minimal TMPRSS11D expression in ducts and acini was confirmed using RNAscope® in situ hybridization (1 independent replication). c, Analysis of available bulk RNA sequencing data suggests that the minor (minor) and parotid (PG) glands are vulnerable to SARS-CoV-2 infection compared to the submandibular glands (SMG); no independent replication. The minor SG express ~3x higher ACE2 compared to the major SG (violin plot highlights the mean with a solid line; minor: 1.42, 0.70; 0.53, respectively). For comparison, the glands express an equivalent amount of TMPRSS2 across all three SG samples (mean 63.60, 49.42; 64.78, respectively). d,e, To further confirm co-expression of ACE2 and TMPRSS2, RNAscope® fluorescent in situ hybridization and immunohistochemistry for pan-cytokeratin (pCK), shows that acini and ducts co-express ACE2 and TMPRSS2 further highlighting their vulnerability to infection in (d) minor and (e) parotid SG (1 independent replication). f, Examples of positive and negative controls for chromogenic assays in Fig. 3 (1 independent replication). g-h, (g) ISH mapping validation pipeline for discovering epithelial expression of SARS-CoV-2 entry factor expression in shedding suprabasal cells, first starting with H&E histochemical staining (1 independent replication). h, ISH was used with negative and positive probe controls. The dotted black box in (g) represents the zoomed-in areas. Experiments using immunofluorescence and in situ hybridization confocal microscopy were completed on tissue sections from 4 separate individuals (2 minor, 2 PG). Curved white arrows in (h) represent direction of differentiated epithelial cell shedding Scale bars: (f-h) 100μm, (a,b,d,e) 50μm.