Figure 7.

Effects of PACAP and/or oxidative stress and/or mechanical stress (MS) on mRNA (A) and protein (B) expression of ADAMTS4 in chondrifying micromass cultures. Optical densities of signals were measured and results were normalized to the optical densities of three-day cultures (C). In panels (A,B), the numbers below the signals represent integrated densities of signals determined by ImageJ freeware. For RT-PCR and Western blot reactions, GAPDH (A) and actin (B) were used as internal controls. Aggrecanase activity (C) was also measured after treatments. Densities and means of three independent experiments (±standard error of the mean) are shown in the figures. Asterisks indicate significant differences compared to the three-day cultures (p < 0.05, one-way ANOVA followed by Tukey’s HSD test). Where ANOVA reported significant differences between the groups (p < 0.05), a post hoc test (multiple comparison versus control group, Tukey’s HSD method) was used to isolate the groups that differed from the control group at p < 0.05. The respective control group was the untreated control when comparison was made between control, H₂O₂-treated, PACAP 1-38-treated, and mechanically stressed groups, whereas the H₂O₂-treated cultures were used as control in the post hoc test when comparison was made between H₂O₂-treated, H₂O₂+PACAP 1-38-treated, and H₂O₂+mechanically stressed samples. * shows significant differences between control cultures and treated samples; ** shows significant differences between single-H₂O₂ treatment and combined treatments; # shows significant differences between single-MS treatment and combined treatments.