1h 2h 4h 6h0

500

1000

1500

2000

2500

3000

3500

4000

serum (-) + DMSO 2 ul

serum (-) + ASA 400 uM

10% FBS (+) + DMSO 2 ul

10% FBS (+) + ASA 400 uM

PG

E2

(pg

/ml)

a b

Procollagen I

serum

aspirin(400 mM)

+ +

+ +

6 h

GAPDH

- -

- -

Figure S1. Serum-up-regulated PGE2 suppresses procollagen I protein expression in fibroblasts. (a) Serum up-regulates PGE2 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 PGE2 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.

Procollagen I

Translationinhibitors

PGE2

(500 nM)

CHX

++

Treatment I (30 min prior to PGE2)

Treatment II (2 h)

GAPDH

CHX Puro 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 PGE2 (500 nM). Translation inhibitors were added first, and 30 min later, PGE2 was added to the indicated wells without changing the medium. 2 h after addition of PGE2, cell lysates were harvested, and collagen I levels in cell lysates were determined by immunoblotting. An immunoblot representative of 2 different experiments is shown.

GAPDH

Cont PGE2 CHX

Figure S3. More uniform inhibition of global protein synthesis by CHX than by PGE2 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 ± PGE2 (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.

PGE2

(500 nM)

p-rpS6

total-rp S6

+ + + + +

1h 2h 4h 8h 24h

- - - - -

a

b

p-rpS6

rpS6

PGE2

(nM)0 1 50 100 500

4h

0

1000

2000

3000

4000

**

rpS

6 p

ho

sph

o t

o t

ota

l rat

io(%

of

Co

ntr

ol)

PGE2

(nM)0 10 50 100 500

Figure S4. PGE2-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 PGE2 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 PGE2 treatment. Data are obtained from 3 different experiments and are expressed as mean SEM. *, P < 0.05 vs control fibroblasts without PGE2 treatment