Figure 1.
WWOX expression in human smokers/e-cigarette–exposed mice and the associated lung endothelial barrier consequences. (A) RNA was extracted from lung tissue harvested from current versus never-/former human smokers. RT-PCR for WWOX (Taqman Assay; Life Technologies) was performed. The bar graph depicts fold-changes in WWOX mRNA expression calculated by the ΔΔCt method with GAPDH as the internal control. Results are shown as means ± SD in n = 3 independent experiments. (B) RNA was extracted from lung tissue harvested from mice exposed to e-cigarette vapor with or without nicotine (control). RT-PCR for WWOX results are shown here. Results are shown as means ± SD in n = 3 independent experiments. The same lung tissues were analyzed by Western blotting for WWOX:actin expression. (C and D) Representative blots are shown. (E) Pulmonary endothelial cells (ECs) were transfected with either a WWOX-targeted or control scrambled siRNA (Dharmacon). A Western blot for WWOX confirms silencing. Transfected ECs were grown to confluency on gold electrodes for ECIS analysis. They were pretreated with a pharmacologic inhibitor of JNK (SP600125) versus vehicle followed by LPS. (F) In separate experiments, ECs were grown in a monolayer on transwell inserts containing 3-micron pores. Then, 10 μg/ml FITC-labeled dextran (3 kD) was added over the cells along with LPS. After 1 hour, media from below the transwell was analyzed by fluorometry for the presence of FITC. Results are shown as means ± SD in n = 3 independent experiments. *P < 0.05, compared with control by Student’s t test. E-cig = electronic-cigarette; ECIS = electrical cell impedance sensing; FITC = fluorescein isothiocyanate; RFU = relative fluorescent unit; si = silencing.