Figure S3.

SARS-CoV-2 co-localizes with the microvilli during later stages of virus infection, related to Figure 3

(A) Two different classes of protrusions on the apical surface of HNEs were observed. Long and wide motile cilia (yellow dashed line) and stubby dome-like microvilli (red dashed line). HNEs (Donor 3, see Table S1) were observed by SEM Arrowhead: microvilli.

(B) Representative double IF staining of SLK and phalloidin in ALI-cultured HNEs from Donor 1, see Table S1.

(C) Representative IF staining of EZR and EBP50 in combination with ACTUB in human nasal tissue.

(D) Representative IF staining of ACTUB and MUC5AC in combination with phalloidin in ALI-cultured HNEs from Donor 1, see Table S1.

(E) SARS-CoV-2-infected HNEs with MOI of 0.3 were stained after 48 hpi. Representative IF staining of either SARS-CoV-2 SP or SARS-CoV-2 NP in combination with phalloidin in SARS-CoV-2-infected HNEs from Donor 1 (see Table S1).

(F) Mock-treated or SARS-CoV-2-infected HNEs with MOI of 0.3 were stained after 6, 24, 48, and 96 hpi. Representative IF staining of SARS-CoV-2 SP, CEP164, and phalloidin in mock or SARS-CoV-2-infected HNEs from Donor 3 (see Table S1) is shown.

(G) Small viral vesicles and large viral-containing vesicles could be observed in the infected cells. SARS-CoV-2-infected HNEs (Donor 4–6, see Table S1) with MOI of 0.3 were fixed after 48 hpi. The cells were observed by TEM. Red arrowhead: small viral vesicle. Yellow arrowhead: large viral-containing vesicle.

(H) IHC staining for SARS-CoV-2 in nasal epithelium of infected K18-hACE2 mice. Nasal epithelium tissues were collected at days 3 post-infection, fixed in 4% PFA, embedded in paraffin wax, and cut into 5-μm sections. Representative IF staining of SARS-CoV-2 SP and ACTUB in combination with Phalloidin in mouse nasal tissue. Scale bars represent 1 μm (A), 5 μm (B-F), and 10 μm (H).