Figure S1.
The phenotype of tracheal MCCs in Daple-KO mice. (A) Analysis of Daple expression using immunoblotting (IB) in WT and Daple-KO MTECs grown at the ALI for 12 d; α-tubulin (αtub) was used as a loading control. (B) Representative images of Odf2 and ZO-1 in WT and Daple-KO tracheas. (C) Analyses of the ratios of total numbers of MCCs against total numbers of apical cells in WT and Daple-KO tracheas (n = 5). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. (D and E) Immunofluorescence images of Daple, Fzd6, and Odf2 (D) or for Daple, Vangl1, and Odf2 (E) in the MCCs of MTECs grown at the ALI for 12 d. Arrows indicate the localization of Daple, Fzd6, and Vangl1. The Odf2 signal (blue) was used as a marker of MCCs. (F) Analysis of mucociliary transport in WT, Daple-KO, and Vangl1-KO adult tracheas using live imaging of fluorescent beads. High-magnification images of the boxed regions are shown in Fig. 1 B. (G) Statistical analyses of Io of BBs at the cell level in WT and Daple-KO tracheas at different developmental stages of E17.5, P2, and adult mice (n = 8–13 cells). Representative images are shown in Fig. 1 G. Two-tailed Mann-Whitney U test; **, P < 0.01; n.s., P ≥ 0.05. (H and I) Statistical analyses of Io values of BBs at the cell level (H; n = 8 cells) and tissue level (I; n = 3 tracheas) in WT, Daple-KO, and Vangl1-KO tracheas. Representative images are shown in Fig. 2 B. Statistical differences were analyzed using two-tailed Mann-Whitney U tests (H) or Kruskal-Wallis tests with Steel-Dwass multiple-comparison tests (I). *, P < 0.05; **, P < 0.01. Scale bars represent 5 µm in B, D, and E and 10 µm in F.

The phenotype of tracheal MCCs in Daple-KO mice. (A) Analysis of Daple expression using immunoblotting (IB) in WT and Daple-KO MTECs grown at the ALI for 12 d; α-tubulin (αtub) was used as a loading control. (B) Representative images of Odf2 and ZO-1 in WT and Daple-KO tracheas. (C) Analyses of the ratios of total numbers of MCCs against total numbers of apical cells in WT and Daple-KO tracheas (n = 5). Two-tailed Mann-Whitney U test; n.s., P ≥ 0.05. (D and E) Immunofluorescence images of Daple, Fzd6, and Odf2 (D) or for Daple, Vangl1, and Odf2 (E) in the MCCs of MTECs grown at the ALI for 12 d. Arrows indicate the localization of Daple, Fzd6, and Vangl1. The Odf2 signal (blue) was used as a marker of MCCs. (F) Analysis of mucociliary transport in WT, Daple-KO, and Vangl1-KO adult tracheas using live imaging of fluorescent beads. High-magnification images of the boxed regions are shown in Fig. 1 B. (G) Statistical analyses of Io of BBs at the cell level in WT and Daple-KO tracheas at different developmental stages of E17.5, P2, and adult mice (n = 8–13 cells). Representative images are shown in Fig. 1 G. Two-tailed Mann-Whitney U test; **, P < 0.01; n.s., P ≥ 0.05. (H and I) Statistical analyses of Io values of BBs at the cell level (H; n = 8 cells) and tissue level (I; n = 3 tracheas) in WT, Daple-KO, and Vangl1-KO tracheas. Representative images are shown in Fig. 2 B. Statistical differences were analyzed using two-tailed Mann-Whitney U tests (H) or Kruskal-Wallis tests with Steel-Dwass multiple-comparison tests (I). *, P < 0.05; **, P < 0.01. Scale bars represent 5 µm in B, D, and E and 10 µm in F.

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