Table 1.
Primers.
Figure 1.
The expression of circadian genes in RA and OA patients.
A. Synovial membrane samples were collected from RA and OA patients (n = 10 for each) and analyzed for circadian gene expression. The relative expression of each gene is expressed as mean ± SD. B. The expression of BMAL1 and PER1 should be in anti-phase. Pearson correlation and the linear fit for the expression of BMAL1 and PER1 in the synovial membrane of RA patients reveals that high expression of BMAL1 correlates with high PER1 expression and vice versa, low BMAL1 expression correlates with low PER1 expression. In contrast to synovium of RA patients, the high expression of BMAL1 was associated with low PER1 expression and conversely, low BMAL1 expression was coupled with high PER1 expression in the synovial membrane of OA patients. Similar findings were observed with NPAS2 and PER1 as well as with ARNTL2 and CRY2. Each dot indicates the Ct value of BMAL1, NPAS2 or ARNTL2 in the x-axis and PER1 or CRY2 in the y-axis in the synovial membrane sample of an individual patient. *p<0.05.
Figure 2.
Localization of BMAL1 protein in RA and OA synovium.
Tissue sections were incubated in rabbit anti-BMAL1 (anti-MOP3). In RA (A and B), relatively strong immunoreactivity was detected in the nuclei (arrows) of the immune cells and fibroblasts, but even stronger immunoreactivity was observed in the cytoplasm of the cells (arrowheads). In contrast to RA, BMAL1 protein was localized only in the nuclei (arrows) of OA synovium (C and D). Irrelevant rabbit IgG was used to confirm the specificity of the BMAL1 antibody (E and F). Scale bar 100 µm.
Figure 3.
Relative clock gene expression over time after serum shocked primary fibroblasts.
Fitted sin graphs of the expression and mean ± SD of the mean from three individual patients per group are shown. The time of serum removal is 0 in the graphs and the relative expression is normalized to the expression before serum (at time = −2). Δb indicates the general difference in the expression i.e., the baseline of the expression. Phase differences are marked as red solid lines and the difference is indicated in hours.
Figure 4.
The oscillation of IL-1β, IL-6 and TNF-α in RA and OA cells after serum shock.
Relative gene expression over time is displayed. Fibroblasts derived from RA synovium display weaker rhythmic expression of IL-1β, IL-6 and TNF-α after clock resetting than cells from OA synovium. Fitted sin graphs of the expression and mean ± SD of the mean from three individual patients per group is shown. The time of serum removal is 0 in the graphs and the relative expression is normalized to the expression before serum (at time = −2). Δb indicates the general difference in the expression i.e., the baseline of the expression. ΔA indicates the differences in the peak amplitude. Phase differences are marked as red solid lines and the difference is indicated in hours.
Figure 5.
TNF-α increased ARNTL2 and NPAS2, and suppresses PER3 and DBP expression but IL-6 did not.
Relative gene expression over time is displayed. Representative data from one experiment is shown. The experiment was repeated five times using fibroblasts from different OA patients. The time of TNF-α or IL-6 addition is 0 in the graphs.