1. PGE 2 (pg/ml) a b Procollagen I serum aspirin (400  M) ++ + + 6 h GAPDH -- - - Figure S1. Serum-up-regulated PGE 2 suppresses procollagen I protein expression in fibroblasts. (a) Serum up-regulates PGE 2 production in fibroblasts. After overnight serum starvation, serum free medium was removed, and cells were incubated in serum-containing medium ± aspirin (400 mM) containing 0.2% DMSO. At indicated time points, supernatants were harvested, and PGE 2 concentration in the supernatants was determined by ELISA. Data obtained from one experiment representative of 2 experiments in which serum and aspirin exhibited the same effect are shown. (b) Serum-stimulated PGE 2 decreases collagen I protein expression. After overnight serum starvation, serum free medium was removed and cells were incubated in serum-containing medium with or without aspirin (400 mM) (or its vehicle DMSO at final concentration 0.2%) for 6 h. Then collagen I protein levels in the cell lysates were determined by immunoblotting. An immunoblot representative of 3 different experiments is shown.

2. Procollagen I Translation inhibitors PGE 2 (500 nM ) CHX + + Treatment I (30 min prior to PGE 2 ) Treatment II (2 h) GAPDH CHX Puro -- + -- - Figure S2. Effect of translation inhibitors on collagen I expression in lung fibroblasts. After overnight serum starvation, cells were cultured in serum-free medium with or without the translation inhibitors cycloheximide (CHX, 10 mM) or puromycin (Puro, 20 mM) and/or PGE 2 (500 nM). Translation inhibitors were added first, and 30 min later, PGE 2 was added to the indicated wells without changing the medium. 2 h after addition of PGE 2, cell lysates were harvested, and collagen I levels in cell lysates were determined by immunoblotting. An immunoblot representative of 2 different experiments is shown.

3. GAPDH ContPGE 2 CHX Figure S3. More uniform inhibition of global protein synthesis by CHX than by PGE 2 in fibroblasts. After overnight serum starvation, normal adult lung fibroblasts were washed once with methionine-free serum-free medium, and then were cultured in methionine-free serum- free medium ± PGE 2 (500 nM) or cycloheximide (CHX, 10 mM) for 30 min. The methionine analog AHA was added at final concentration 25 mM into each well, and cells were further incubated with AHA for 40 min. Then, cell lysates were harvested, and Click-iT Reaction® was performed on cell lysates to conjugate biotin to AHA analog incorporated into newly synthesized proteins. After electrophoresis of cell lysates, newly synthesized proteins containing biotin-conjugated AHA were detected with streptavidin-HRP. An immunoblot representative of 5 different experiments is shown.

4. PGE 2 (500 nM) p-rpS6 total-rp S6 +++++ 1h2h 4h8h 24h ----- a b p-rpS6 rpS6 PGE 2 (nM) 0 150 100 500 4h PGE 2 (nM) 01050100 500 Figure S4. PGE 2 -enhanced phosphorylation of rpS6 is dose-dependent and lasts for more than 8 h. After overnight serum starvation, cell culture medium was changed to fresh serum-free medium, and normal adult lung fibroblasts were then incubated with or without PGE 2 at various concentrations (a) or at 500 nM (b). At 4h (a) or indicated time points (b), cell lysates were harvested, and levels of total and phosphorylated rpS6 in cell lysates were determined by immunoblotting. An immunoblot representative of 3 (a) or 2 (b) different experiments is shown. In a, right, densitometric ratio of phospho:total rpS6 at each concentration was expressed relative to that in control fibroblasts without PGE 2 treatment. Data are obtained from 3 different experiments and are expressed as mean  SEM. *, P < 0.05 vs control fibroblasts without PGE 2 treatment