Decreased bleomycin-induced apoptosis in hTERT transfected MLE cells.

<p>(A) PCR-based telomerase activity in Mouse-Lung Epithelial (MLE) cells transfected with hTERT⁺ or control hTERT (hTERT^ctrl) expression vectors. hTERT⁺ and hTERT^ctrl MLE cells were exposed to bleomycin (0.06mU) or control saline. (B) Flow cytometry analysis demonstrating decreased Annexin V staining in bleomycin-treated hTERT⁺ compared to hTERT^ctrl cells. (C) Bar diagram representing the fold ratio (bleomycin/saline) of MLE cell apoptosis in hTERT⁺ vs. hTERT^ctrl MLE cells (n = 4, *p<0.05).</p

Fas expression is unchanged in hTERT⁺-transfected MLE cells.

<p>(A) Flow cytometry analysis (histogram and dot plots) showing similar Fas expression in hTERT⁺ vs. hTERT^ctrl MLE cells. (B) Graphic presentation of FACS analysis from three independent experiments (n = 4).</p

Decreased Fas-induced apoptosis in hTERT⁺ vs. hTERT^ctrl transfected MLE cells.

<p>MLE cells were transfected with hTERT or control expression vector, and exposed to Fas or control IgG mAb (10 μg/ml, 24h). (A) Histogram plot and (B) confocal microscopy of Annexin V staining (blue) and PI (red) in hTERT⁺ vs. hTERT^ctrl cells showing decreased Annexin V staining in hTERT⁺ when compared to hTERT^ctrl transfected cells (MLE). Inserted numbers represent the percentage of Annexin V-stained cells (blue) among total cells in the field (red) with standard deviation (SD). 10–15 fields were counted. (C) Western blots of caspase-8 cleavage into p42 and p18 subunits. Cleaved/uncleaved-caspase-8 optical density (OD) ratios are presented, showing decreased caspase 8 cleavage in hTERT⁺ when compared to the hTERT^ctrl-transfected MLE cell line. Representative results of two different experiments with similar results.</p

Downregulation of FLIP levels annuls hTERT-mediated resistance to Fas-induced apoptosis in human lung epithelial A549 cells.

<p>(A) Western blot of FLIP expression using anti-FLIP mAb in an hTERT+shRNA^Ctrl (sh-scrambled/control) vs. an hTERT+shFLIP-RNA transfected-A549 cell line. OD ratios show decreased FLIP expression in hTERT⁺ A549 cells following further transfection with shFLIP when compared to those that were further transfected with control shRNA. (B) Histogram plot of Annexin V staining and (B) Western blot of caspase 3 cleavage in hTERT⁺ human lung epithelial A549 cells after infection with (hTERTshCtrl) and hFLIP-RNA lentiviral vector (hTERTshFLIP) and subjection to agonist Fas DX2 mAb. Representative results of two different experiments for each assay, with similar results showing increased apoptosis in hTERT⁺ A549 cells transfected with shFLIP-RNA.</p

Apoptotic gene expression in MLE cells after bleomycin treatment.

<p>(A) Specific cDNA fragments of 96 apoptosis-related genes were hybridized with cDNA probes synthesized from two total RNA samples corresponding to untreated (Control) mouse lung epithelial cells and epithelial cells treated with 0.06mU of bleomycin for 72h (BLEO). (B) Relative expression levels of genes relevant to apoptosis. Among other survival genes, FLIP was shown to be downregulated after exposure to bleomycin. The degree of gene expression after bleomycin-and saline-control treatment, as indicated by fold changes, was calculated by raw densitometry values by comparing signal intensity to RPLA 13A and then quantified by densitometry after background subtraction and determined as OD.</p

hTERT⁺-transfected human lung epithelial A549 cells upregulate FLIP levels and acquire resistance to Fas induced apoptosis.

<p>(A) Western blot of FLIP expression using anti-FLIP mAb in hTERT^ctrl (control) vs. hTERT⁺ transfected cells from a human lung epithelial cell line (A549). Optical densities showing increased FLIP in hTERT⁺ human lung A549 epithelial transfected cells are presented (OD). (B) Histogram plots of flow cytometry analysis showing decreased Annexin V staining in hTERT⁺ (red) when compared to hTERT^ctrl (black) transfected cells (A549). (C) Western blot showing decreased caspase 3 cleavage in hTERT⁺, when compared to the hTERT^ctrl transfected A549 cell line. Cleaved/uncleaved caspase 3 OD ratios are presented. Representative results of two different experiments for each assay, with similar results.</p

FLIP is upregulated in hTERT⁺ vs. hTERT^ctrl transfected MLE cells.

<p>(A) Flow cytometry dot plots of FLIP expression, and (B) Western blot using anti-FLIP mAb in hTERT⁺ vs. hTERT^ctrl (control) transfected cells. OD ratios showing increased FLIP in hTERT⁺ mouse lung MLE epithelial transfected cells are presented. Representative results of two different experiments for each assay, with similar results.</p