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. 2012 Oct 5;91(4):685–693. doi: 10.1016/j.ajhg.2012.08.022

Figure 3.

Figure 3

HEATR2 Localization in Ciliated Airway Epithelial Cells

(A and B) Photomicrographs of normal human lung section (scale bar = 100 μm) (A) and bronchial epithelium (B) following immunofluorescent staining for HEATR2, which reveals the cytoplasmic localization of the protein (HEATR2, red) only in ciliated cells (cilia marker, acetylated α-tubulin (α-tub), green; nuclei stained with DAPI, blue) (scale bar = 10 μm).

(C and D) Immunofluorescent staining of nasal epithelial cells cultured at an air-liquid interface from a healthy subject (NL) showing the presence of HEATR2 (C) and an individual with PCD showing a reduction of HEATR2 levels (D) (scale bar = 5 μm).

(E) Immunoblot analysis of tracheobronchial (TR) epithelial cells and nasal epithelia (NP) cells from a healthy subject and an individual with PCD (PCD) confirms the very low level of the mutant form of HEATR2.

(F and G) Immunoflourescent staining in representative human airway epithelial cells transfected with nontargeted, scrambled shRNA sequences (NT-shRNA) (F) and HEATR2-specific shRNA sequences (G) for HEATR2 (red) and acetylated α-tubulin (green) (scale bar = 5 μm).

(H) Immunoblot analyses of airway epithelial cells transfected with each of three different HEATR2-specific shRNA (1–3) or nontargeted shRNA (NT) sequences and nontransfected control cells (M).

(I–K) Ultrastructural appearance of cilium from the proband (I), and cilia from airway epithelial cells following transfection with (J) nontargeted and (K) HEATR2-targeted shRNA sequence 1. Blue and red arrows indicate inner and outer dynein arms, respectively.