Figure 4

Inhibitory role of AIM in cigarette smoke extract–induced apoptosis in U937 cells. (A) Fluorescence microscopic detection of nuclear staining with Hoechst 33258: control (left panel), CSE (5.0%) treated (middle panel), and CSE (5.0%) treated in the presence of conditioned medium containing AIM (right panel). Lower panel shows the percentage (±SEM) of apoptotic cells from three independent experiments. *p < 0.05. (B) DNA fragmentation assay with or without CSE (1.0 or 5.0%) treatment in the absence or presence of conditioned medium containing AIM. (C) Measurement of DNA contents by flow cytometric analysis with propidium iodide (PI) staining. Shown is the relative percentage (±SEM) of hypodiploid apoptotic cells compared to control treated cells from four independent experiments. *p < 0.05. (D) Western blotting (WB) using anti-caspase-9, anti-caspase-8, and anti-β-actin in control treated (lane 1), CSE (5.0%) treated (lane 2), and CSE (5.0%) treated in the presence of conditioned medium containing AIM (lane 3). Protein samples were collected after 16 h treatment. Shown is a representative experiment of 3 showing similar results. (E) Fluorescence microscopic detection of nuclear staining with Hoechst 33258: control siRNA transfected (left panel) and BcL-xL siRNA transfected (right panel) cells were treated CSE (5.0%) in the presence of conditioned medium containing AIM. Lower panel shows the percentage (±SEM) of apoptotic cells from three independent experiments. Open bar is control siRNA transfected and filled bar is BcL-xL siRNA transfected. *p < 0.05. (F) Measurement of DNA contents by flow cytometric analysis with PI staining. Shown is the relative percentage (±SEM) of hypodiploid apoptotic cells compared to control treated cells from three independent experiments. Open bar is control siRNA transfected, filled bar is BcL-xL siRNA transfected, and horizontal crosshatched bar is BcL-xL siRNA transfected with conditioned medium containing AIM. *p < 0.05.