Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine
Figure 3
Effect of LV transduction on occludin localization. Expression of occludin mRNA by RT-PCR (a). Note that occludin (OCLN) transcript is detected in 16HBE14o- cells (lane 1) but not in PBMCs (lane 2). -actin (ACT) transcripts were detected in 16HBE14o- and PMBCs (lanes 4 and 5, respectively). In lane 3, HindIII marker. Polarized 16HBE41o- cells were incubated with 50 (C), 500 (D) or 2000 (E) MOIs and, 24 hours later, occludin localization was evaluated by immunofluorescence and confocal microscopy. Controls included mock cells (B) and cells treated with 12 mM EGTA for 30 minutes (F). En-face micrographs are shown. Occludin-specific signal is in green. A peripheral chicken-wire pattern of occludin localization is noted in B and C. In D and E, white arrows point out to lack of occludin staining at cell borders and between cells. In F, occludin staining is lost from cell borders and distributed intracellularly. Note that some intracellular signal is visible also in B-E. Bar: 10 m.
