Impact of cell density on SP-B mRNA expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated (<b>A</b>, <b>D</b>) or incubated with 1 μM dexamethasone for 48 h (<b>B</b>, <b>E</b>). SP-B mRNA levels were normalized to β-actin and fold differences compared to 0.5 × 10⁴ untreated cells per cm² were calculated (<b>A</b>, <b>B</b>). Means +SD of n = 4 independent experiments are shown. (<b>C</b>) C_T range achieved for β-actin and SP-B mRNA combined for all different cell quantities with and without dexamethasone treatment. (<b>D</b>, <b>E</b>) Representative bright field images of H441 cells at different quantities without (<b>D</b>) and with (<b>E</b>) dexamethasone treatment. The bar in the upper left image represents 500 μm. *** <i>p</i> < 0.001 compared to 0.5 × 10⁴ cells per cm², ### <i>p</i> < 0.001 compared to 1 × 10⁴ cells per cm², $$ <i>p</i> < 0.0 1 and $$ $ <i>p</i> < 0.001 compared to 2.5 × 10⁴ cells per cm², §§ <i>p</i> < 0.01 and §§§ <i>p</i> < 0.001 compared to 5 × 10⁴ cells per cm².</p

Impact of cell density on mature SP-B expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone. After 48 h immunoblotting against SP-B and β-actin was performed. SP-B levels were normalized to β-actin and fold differences compared to 2.5 × 10⁴ dexamethasone-treated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. ** <i>p</i> < 0.01 compared to 5 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

SP-B mRNA expression in H441 cells co-cultured with A549 cells at different cell densities.

<p>Different quantities of H441 cells were seeded alone or co-cultured with different quantities of A549 cells as indicated for 48 h. (<b>A</b>) SP-B mRNA levels were normalized to β-actin and fold differences compared to 1 × 10⁴ H441 cells per cm² without A549 cells were calculated. Means +SD of n = 4 independent experiments are shown. (<b>B</b>, <b>C</b>) Representative bright field images of H441 cells alone (<b>B</b>) or co-cultured with A549 cells (<b>C</b>). The bar in the upper left image represents 500 μm. *** <i>p</i> < 0.001 compared to 1 × 10⁴ H441 cells per cm² without A549 cells, ### <i>p</i> < 0.001 compared to 2 × 10⁴ H441 cells per cm² without A549 cells, $<i>p</i> < 0.05 and$ $$ <i>p</i> < 0.001 compared to 7.5 × 10⁴ H441 cells per cm² without A549 cells, §§ <i>p</i> < 0.01 compared to 2 × 10⁴ H441 cells per cm² co-cultured with 1 × 10⁴ A549 cells per cm².</p

Immunoblots against claudin-5 and claudin-8 from H441 cells at different densities.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) quantities and either left untreated or incubated with 1 μM dexamethasone for 48 h. Immunoblots against claudin-5 (<b>A</b>) and claudin-8 (<b>B</b>) were performed, protein levels were normalized to total blotted protein, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. * <i>p</i> < 0.05 compared to 1 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

Impact of cell density on mature SP-B expression in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone. After 48 h immunoblotting against SP-B and β-actin was performed. SP-B levels were normalized to β-actin and fold differences compared to 2.5 × 10⁴ dexamethasone-treated cells per cm² were calculated. Means +SD of n = 3 independent experiments are shown. ** <i>p</i> < 0.01 compared to 5 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm².</p

Impact of cell density on the expression of SP-B transcription factor mRNAs in H441 cells.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) densities and either left untreated or incubated with 1 μM dexamethasone for 24 h. qPCR of TTF-1 (<b>A</b>), Sp1 (<b>B</b>), Sp3 (<b>C</b>), HNF-3α (<b>D</b>), and ErbB4 mRNA (<b>E</b>) was performed, mRNA levels were normalized to those of GAPDH, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 5 experiments are shown. *** <i>p</i> < 0.001 compared to 1 × 10⁴ untreated cells per cm², ### <i>p</i> < 0.001 compared to 1 × 10⁴ dexamethasone-treated cells per cm², $$ $ <i>p</i> < 0.001 compared to 7.5 × 10⁴ untreated cells per cm².</p

SP-B mRNA expression in H441 cells co-cultured with A549 cells at different cell densities.

<p>Different quantities of H441 cells were seeded alone or co-cultured with different quantities of A549 cells as indicated for 48 h. (<b>A</b>) SP-B mRNA levels were normalized to β-actin and fold differences compared to 1 × 10⁴ H441 cells per cm² without A549 cells were calculated. Means +SD of n = 4 independent experiments are shown. (<b>B</b>, <b>C</b>) Representative bright field images of H441 cells alone (<b>B</b>) or co-cultured with A549 cells (<b>C</b>). The bar in the upper left image represents 500 μm. *** <i>p</i> < 0.001 compared to 1 × 10⁴ H441 cells per cm² without A549 cells, ### <i>p</i> < 0.001 compared to 2 × 10⁴ H441 cells per cm² without A549 cells, $<i>p</i> < 0.05 and$ $$ <i>p</i> < 0.001 compared to 7.5 × 10⁴ H441 cells per cm² without A549 cells, §§ <i>p</i> < 0.01 compared to 2 × 10⁴ H441 cells per cm² co-cultured with 1 × 10⁴ A549 cells per cm².</p

Influence of cell density on SP-B mRNA stability in H441 cells.

<p>Different quantities of H441 cells were seeded as indicated and either left untreated or incubated with 1 μM dexamethasone and/or 10 μg/mL actinomycin D for 24 h. SP-B mRNA levels were normalized to β-actin and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated for each group. Means +SD of n = 4 experiments are shown. Statistical analyses were performed using Student’s unpaired <i>t</i> test and only relevant significances are shown. ** <i>p</i> < 0.01 and *** <i>p</i> < 0.001 compared to 1 × 10⁴ untreated cells per cm², ## <i>p</i> < 0.01 and ### <i>p</i> < 0.001 compared to 1 × 10⁴ dexamethasone-treated cells per cm², $$ $ <i>p</i> < 0.001 compared to 7.5 × 10⁴ untreated cells per cm², §§§ <i>p</i> < 0.001 compared to 7.5 × 10⁴ dexamethasone-treated cells per cm², †† <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone- and actinomycin D-treated cells per cm².</p

Primers for qPCR.

<p>Primers for qPCR.</p

Influence of cell density on mRNA expression of tight junction or tight junction-related proteins in H441 cells.

<p>H441 cells were seeded at low (1 × 10⁴ cells per cm²) and high (7.5 × 10⁴ cells per cm²) densities and either left untreated or incubated with 1 μM dexamethasone for 24 h. qPCR of claudin-1 (<b>A</b>), claudin-2 (<b>B</b>), claudin-3 (<b>C</b>), claudin-4 (<b>D</b>), claudin-5 (<b>E</b>), claudin-7 (<b>F</b>), claudin-8 (<b>G</b>), occludin (<b>H</b>), and ZO-1 mRNA (<b>I</b>) was performed, mRNA levels were normalized to those of GAPDH, and fold differences compared to 1 × 10⁴ untreated cells per cm² were calculated. Means +SD of n = 5 experiments are shown. * <i>p</i> < 0.05 and ** <i>p</i> < 0.01 compared to 1 × 10⁴ untreated cells per cm², # <i>p</i> < 0.05 and ## <i>p</i> < 0.01 compared to 1 × 10⁴ dexamethasone-treated cells per cm², $<i>p</i> < 0.05 and$ $ <i>p</i> < 0.01 compared to 7.5 × 10⁴ untreated cells per cm².</p