Figure 4
From: Somatic cell hemoglobin modulates nitrogen oxide metabolism in the human airway epithelium
Both hemoglobin β overexpression and hemoglobin β knockdown decrease ALI ciliation. (A) RT-PCR analysis of Hbβ mRNA expression in Day 33 ALI primary human bronchial epithelial cells at ALI, transduced with empty vector (Hbβ Ctrl), Hemoglobin β over-expressed vector (Hbβ O.E.), Scrambled Ctrl (SH Ctrl), and Hemoglobin β knockdown shRNA ‘s 1-4 (SH1-4). (B) Representative AB-PAS staining of paraffin embedded sections of Day 33 ALI cultures from the Hbβ under- and over-expressing, and control, cells as described in (A). 20 × magnification. Arrow shows the membrane thickness that is equal 20 μm. These images were read by three blinded observers each, counting the number of ciliated cells as a fraction of cells on the apex of the culture (see C). (C) Percentage of ciliated apical cells were quantitated blindly by three independent investigators. Cilia were absent from the Hbβ over-expressing cells. In the Hbβ knockdown cells, ciliary expression was decreased relative to wt and scrambled shRNA expressing cells, shown in aggregate (Purple is Sh1, Red is Sh2, Green is Sh3, and Blue is Sh4), (*p = 0.041).
