Figure 1.

Cell density modulates COX-2 induction. A and B, cell density modulates the induction of COX-2 at the protein level. H358 cells (about 2 × 10⁵ cells) were seeded onto 10-, 6-, and 3.5-cm plates for low, medium, or high cell densities, respectively, for 6 h and then treated with 10 ng/ml IL-1β (A) or 10 ng/ml TNFα (B) for the times indicated. Cells were then harvested and analyzed by immunoblotting (IB) with the indicated antibodies. C, cell density modulates the induction of COX-2 at the transcriptional level. H358 cells (about 2 × 10⁵ cells) were seeded onto 10-, 6-, and 3.5-cm plates for different cell densities and then treated with IL-1β for the times indicated after 6 h. Cells were then harvested for RNA extraction. The mRNA level of COX-2 was measured by qRT-PCR. n = 3 independent experiments. Data are presented as mean ± S.D. Error bars represent S.D. *, p < 0.05; **, p < 0.01; and ***, p < 0.001, by unpaired, two-tailed Student's t test. D and E, cell density modulates COX-2 induction. H358 cells were cultured on cover glasses in 24-well plates at different densities (2.5 × 10⁴, 7.5 × 10⁴, and 2.25 × 10⁵ cells/well) for 6 h, stimulated with IL-1β for another 2 h, and fixed for immunofluorescence (IFC) staining for COX-2. Nuclei were counterstained with Hoechst (D). Relative mean fluorescence intensities of COX-2 of each cell were determined (E). n = 3 independent experiments. Data are presented as mean ± S.D. Error bars represent S.D. **, p < 0.01, by unpaired, two-tailed Student's t test (E).