Figure 4.

Effect of interleukin-1-beta (IL-1β) on lipocalin-type prostaglandin D synthase (L-PGDS) expression in osteoarthritis chondrocytes. Chondrocytes were treated with 100 pg/mL IL-1β for the indicated time periods or with increasing concentrations of IL-1β for 48 hours. (a, b) Total RNA was isolated and reverse-transcribed into cDNA, and L-PGDS and GAPDH mRNAs were quantified using real-time polymerase chain reaction. All experiments were performed in triplicate, and negative controls without template RNA were included in each experiment. Results are expressed as fold changes, considering 1 as the value of untreated cells, and represent the mean ± standard error of the mean (SEM) of four independent experiments. *P < 0.05 compared with unstimulated cells. (c, d) Cell lysates were prepared and analyzed for L-PGDS and β-actin proteins by Western blotting. Representative Western blots are shown in the upper panels. In the lower panels, the bands were scanned, and the L-PGDS band intensity values were normalized to the corresponding β-actin band intensity value. Data are expressed as fold induction, considering 1 as the value of unstimulated cells, and represent the mean ± SEM of four independent experiments. *P < 0.05 compared with unstimulated cells. (e, f) Conditioned media was collected and analyzed for prostaglandin D₂ (PGD₂) content. Results are expressed as the mean ± SEM of four independent experiments. *P < 0.05 compared with unstimulated cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.