Surfactant proteins in absence or presence of indomethacin alone or in combination with PGE2.

<p>A) AECII were cultured without or with the cyclooxygenase inhibitor indomethacin (1 µM) and SP-A1, SP-A2; SP-B; SP-C and SP-D mRNA were measured by real-time PCR after culture. Relative Expression of surfactant proteins in cultures without indomethacin (C, hatched bar) were used to calculate the percentage of remaining surfactant expression after cyclooxygenase inhibition (indomethacin, n = 5)). B) Change of relative expression of surfactant proteins in cultures without (C; left panel) and with inhibition of cyclooxygenase by indomethacin (1 µM) and substitution with external PGE2 (10 nM, right panel) measured by real-time PCR. Bar charts show mean ± SD (surfactant proteins n = 8; rE = relative expression; c = control; PGE2 = prostaglandin E2).</p

SP-B, C, and D mRNA level in presence of indomethacin and PGE2.

<p>Freshly isolated AEC-II were cultured for 24 h with indomethacin/PGE2 and the indicated substances. After the culture period SP-B (A), SP-C (B) and SP-D (C) mRNA expression was measured by real-time PCR. Due to the large variation in surfactant protein mRNA expression cultures were separated according their basal median expression of the respective surfactant protein (low level SP-B<1088, SP-C<970059, SP-D<11515 black bars; high level SP-B>1088, SP-C>970059, SP-D>11515 hatched bars; A, B, C). Bar charts show means ± SD (n_low = 6, n_high = 5, A, B,; n_low = 6, n_high =6, C; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide; PGE2 = prostaglandin E2).</p

Western blot analysis of total cellular lysate of SP-A protein.

<p>Equal numbers of freshly isolated AEC-II (2×10⁶cells/well) were incubated with Brefeldin A 1 M for 15 min and stimulated with dbcAMP (1 mM), roflumilast-N-oxide at 1 µM or 3 nM. Total cell lysates were separated at reducing conditions using 12% SDS-PAGE and stained with anti-SP-A, anti-SP-B and SP-C. A: representative blot for low (left panel) and high (right panel) SP-A producer, purified SP-A from a patient with alveolar proteinosis was used as positive control, B: increase in SP-A protein expression in low SPA producers measured by densitometric analysis of the blots. Values are expressed as percentage of non-stimulated cells (n = 6), C: representative blot for low (left panel) and high (right panel) SP-B and SP-C producer. (c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide; + = positive control, purified SP-A).</p

PDE1-5 activities in human AEC-II cells.

<p>Cell lysates of human AEC-II probed for PDE1-5 activities at 0.5 µM cAMP or cGMP substrate concentrations as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038369#s2" target="_blank">Methods</a>. PDE1 was measured with cAMP or cGMP as substrate. Results are shown as mean ±SEM from three different donors.</p

SP-A1 and SP-A2 mRNA level with indomethacin and PGE2.

<p>Cells were cultured for 24 h in presence of indomethacin (1 µM), PGE2 (10 nM) and stimulated with dbcAMP (1 mM) or roflumilast-N-oxide (3 nM or 1 µM; A, B). Cultures were split according their basal median expression of SP-A1/SP-A2 (low level SP-A1<136, SP-A2<474, black bars; high level SP-A1>136, SP-A2>474, hatched bars; C, D). Values presented are means ± SD (n = 19; A,B; n_high = 9, n_low = 10, C, D; rE = relative expression; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide, PGE2 = prostaglandin E2).</p

SPA-1 and SP-A2 mRNA level.

<p>Freshly isolated AEC-II were cultured for 24 h with or without the indicated substances. After the culture period expression of SP-A1 (A) and SP-A2 (B) mRNA was measured by real-time PCR. Due to the large variation in SP-A expression cultures were grouped according their basal median expression of SP-A1/SP-A2 (low level SP-A1<1436, SP-A2<2500, black bars; high level SP-A1>1436 and SP-A2>2500, hatched bars; C, D). Bar charts show mean±SD (n = 13, A; n = 13, B; n_high = 6, n_low = 7, C; n_high = 7, n_low = 6, D; rE = relative expression; c = non-stimulated control; dbcAMP = dibutyryl-cAMP; RNO = roflumilast-N-oxide).</p

Primer sequences.

<p>Primer sequences.</p