Anti α smooth muscle actin

To induce primary cilia formation, fibroblasts were grown in serum-reduced media for 48 h. The RPE cells were grown on Matrigel-coated Lab-Tek 8-well chamber slides (Thermo Fisher Scientific #177402). The cells were subsequently fixed with 4% paraformaldehyde (Hounisen, Midtjylland, Denmark) for 15 min, permeabilized with 0.2% Triton X-10 in PBS and blocked with 3% bovine serum albumin (BSA) in permeabilization buffer. Incubation with primary antibodies diluted in 3% BSA was done for 45 min at room temperature or overnight at 4 °C. Secondary antibody incubation was carried out for 1 h at room temperature before mounting. Nuclei were visualized using DAPI. All images were obtained using an Olympus Fluoview 1000 confocal microscope and images were analyzed using ImageJ. The following antibodies were used: Anti-α-Acetylated tubulin (Sigma-Aldrich #T6793, St. Louis, MO, USA), Anti-Smoothened (Abcam #ab38686, Cambridge, UK), Anti-ARL13B (Proteintech #1711-1-AP, Rosemont, IL, USA), anti-α-smooth muscle actin (SMA) (Dako #M0851), anti-α-fetoprotein (AFP) (Dako #A0008), anti-βIII tubulin (βtub) (Sigma-Aldrich #T8660) and anti-ZO-1 (Thermo Fisher Scientific #40-2200).

The airway biopsies used in this study were obtained from subjects recruited from the Melbourne Epidemiological Study of Childhood Asthma (MESCA) cohort (all aged 42 years at the time of biopsy) (Ward et al., 2008 (link); Qiao et al., 2017 (link); Li et al., 2019 (link)). Asthma severity in this study was classified by using contemporary Global initiative for Asthma guidelines as described in detail in (Ward et al., 2008 (link)). The demographic data of the donors are provided in Supplementary Table S4. Paraffin-embedded sections of human airways and ALI cultures differentiated from NHBE cells were stained with ACE2 (Abcam) using three-layer immunoperoxidase protocol. ACE2 expression level was determined by a semi-quantitative IHC scoring method. In parallel with ACE2 staining, selected sections were stained with anti-α-smooth muscle actin (Dako, 1:400) as positive control and isotype IgG as negative control. The ACE2 staining was qualitatively scored from 0 to 5 by an experienced operator and confirmed by an additional operator (each blinded to group allocations), with 0 denoting no staining and 5 indicating heavy uniform and extensive staining (Supplementary Figure S4).