Figure 3. Effect of LC3 and ATG5 knock down on CSE-induced cell senescence in HBEC. (A) RT-PCR (upper panel), using primers to LC3B and β-actin, was performed from total RNA harvested from control siRNA (lane 1) and LC3B siRNA (lane 2) transfected HBEC after 48 h incubation. WB (lower panel) using LC3 and anti- β-actin of cell lysates from control siRNA (lane 1) and LC3 siRNA (lane 2) transfected HBEC after 48 h in the presence of protease inhibitors (E64d 10 μg/ml and pepstatin A10 μg/ml). (B) Senescence associated β-galactosidase (SA-β-gal) staining of control or LC3B siRNA transfected HBEC. Shown in panel is the percentage (± SEM) of SA-β-gal positive cells from three independent experiments. Open bar is no treatment and filled bar is CSE (1.0% for 48 h) treated and horizontal crosshatched bar is CSE (2.5% for 48 h) treated. *p < 0.05. (C) western blot (WB) using anti-p21 or β-actin in control or LC3B siRNA transfected HBEC. HBEC were treated with control (lane 1, 4), CSE (1.0%) (lane 2, 5), and CSE (2.5%) (lane 3, 6). Protein samples were collected after 48 h treatment with CSE. Shown is a representative experiment of 3 showing similar results. (D) western blot (WB) using ATG5 and anti- β-actin of cell lysates from control siRNA (lane 1) and ATG5 siRNA (lane 2) transfected HBEC after 48 h. (E) Senescence associated β-galactosidase (SA-β-gal) staining of control or ATG5 siRNA transfected HBEC. Shown in panel is the percentage (± SEM) of SA-β-gal positive cells from three independent experiments. Open bar is no treatment and filled bar is CSE (1.0% for 48 h) treated. *p < 0.05. (F) western blot (WB) using anti-p21 or β-actin in control or ATG5 siRNA transfected HBEC. HBEC were treated with control (lanes 1 and 4), and CSE (1.0%) (lanes 2 and 4). Protein samples were collected after 48 h treatment with CSE. Shown is a representative experiment of three similar results.