Figure 1.

Heat shock (HS) augments extrinsic apoptosis. (A) Mouse lung epithelial (MLE) cell line 15 (MLE15) cells were incubated with indicated concentration of recombinant mouse TNF-α for 24 hours at 37°C with or without a concurrent 2-hour 42°C HS, and survival was assessed by crystal violet staining residual adherent cells and measuring absorbance at 570 nm. Cell death was different between HS and no HS cells (P < 0.05 by MANOVA). (B) MLE15 cells were incubated with or without 0.3 ng/ml TNF-α for 24 hours at 37°C with 2-hour HS beginning at the indicated time after TNF-α; 37°C indicates no HS. (C) MLE15 cells were incubated for the indicated time at 37°C or 42°C with or without 2 ng/ml TNF-α, lysed, and immunoblotted for active caspase-3 (C3), poly ADP-ribose polymerase (PARP), and β-tubulin. PARP-CL, cleaved PARP; PARP-FL, full-length PARP. (D) MLE15 cells were treated as in (B), but with 2.5 μg/ml Jo2 anti-Fas antibody. (E and F) MLE15 cells were preconditioned with 2-hour HS beginning at the indicated time before induction of apoptosis with 0.3 ng/ml TNF-α (E) or 2.5 μg/ml Jo2 antibody (F), with or without concurrent 2-hour HS, and 24-hour survival assessed by crystal violet staining. (G) MLE15 cells without (left panel) or with 2-hour HS preconditioning beginning 16 hours before a 2-hour incubation at 37°C or 42°C, with or without 2 ng/ml TNF-α, were lysed and immunoblotted for active caspase-3 and PARP. All graphs show means (±SE) of six experiments. (B and D) *P < 0.05 versus TNF-α or Jo2 without HS. (E and F) ^†P < 0.05 and ^¶P < 0.05 versus similarly preconditioned and treated cells without concurrent HS and cells that were sham (37°C) preconditioned. A570, absorbance at 570 nm.