Extended Data Figure 4. miR-34/449 deficiency causes defective ciliation and basal body docking in mouse airway MCCsa. miR-34/449 TKO trachea exhibit reduced MCC ciliation. Quantification of fully ciliated MCCs (γ-tub and Ac-α-tub double-positive) and partially/non-ciliated MCCs, Ac-α-tub weak/negative) was performed in littermate controlled DKO and TKO mouse tracheas, using data from all three experiments in Figure 3a. The number of cells with MCC identity (γ-tub positive) is unaffected in TKO tracheas, yet one third of the TKO MCCs display aberrant Ac-α-tub staining, indicating ciliation defects. Paired t-test, ns: P > 0.05, *** P < 0.001. b. The mir-34a-/-; mir-34b/34c-/- DKO mice exhibit normal ciliogenesis in tracheal MCCs. Whole tracheas from age matched adult wild-type and mir-34a-/-; mir-34b/34c−/−- DKO mice were analyzed by immunofluorescence staining for Ac-α-tub (cilia) and γ-tubulin (basal bodies). n=3 c. miR-34/449 TKO primary tracheal epithelial cells exhibit ciliation defects in air liquid interface (ALI) culture. ALI culture of MCCs were derived from tracheas of littermate-controlled mir-34a-/-; mir-34b/34c+/-; mir-449-/- and TKO mice, and subjected to immunofluorescence staining for Ac-α-tub (cilia) and γ-tub (basal bodies). In TKO and control ALI culture, comparable levels of γ-tub positive cells are observed, yet a large portion of TKO γ-tub positive cells displayed a partial or complete loss of Ac-α-tub staining. a: fully, b: partially, c: non ciliated MCCs; n=2. d. Basal bodies fail to dock to the apical membrane of miR-34/449 TKO MCCs in ALI culture. Lateral projections of confocal micrographs described in (c) show impaired apical localization of γ-tub staining in TKO MCCs from ALI cultures, suggesting a defective basal body docking to the apical membrane. e. mir-34a-/-; mir-449-/- DKO trachea exhibit no defects in basal body docking using transmission electron microscopy (TEM) analyses. n=3. f. TKO tracheal MCCs exhibit a defective subapical Actin network. Whole tracheas from adult wild-type, mir-34a-/-; mir-34b/34c-/- DKO and TKO mice were analyzed by immunofluorescence staining for Ac-α-tub (cilia) and phalloidin-488 (Actin). n=2. All error bars represent s.e.m.
Image published in: Song R et al. (2014)
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