Fig 2: Distribution of CDH6 protein in RA synovial tissue using confocal microscopy. Representative images of RA synovium stained with anti-CDH6 and -CD68 (macrophage) antibodies demonstrate the lining and sublining expression of CDH6 (type A and B synoviocytes), most prominently in lining FLS (solid white arrow) and macrophages (open arrows) (n = 4 separate tissues were stained). CDH6 and CD68 are also expressed in the sublining region. Images were taken with Olympus VS200 slide scanner (20x) high resolution. S4 Figure shows the negative controls for these images (no primary antibody or control rabbit IgG, and control mouse IgG)

Fig 3: CDH6 regulates RA FLS migration. (A) Effect of CDH6 knockdown on FLS migration. Right side: representative wound healing assay of RA FLS transfected with CDH6 siRNA (siCDH6) or scramble control (CT) with or without PDGF-stimulation (10 ng/ml for 24 h). Left side: Graphs represent the percentage of migration inhibition induced by siCDH6 compared to CT in both RA and OA FLS (RA: n = 6 and OA: n = 4). (B) Effect of Anti-CDH6 on RA FLS migration. Right side: representative migration assay with or without PDGF stimulation in RA FLS (10 ng/ml for 24 h). Cultures included 2ug/ml of anti-CDH6 antibody or anti-IgG antibody. Left side: Quantification of cells that migrate into the wound after 24 h of PDGF stimulation (n = 6). All data were presented as mean ± SEM. *P ≤ 0.05, ANOVA multiple comparison test

Fig 4: Regulation of CDH6 in FLS. (A) CDH6 was measured in OA and RA FLS by qRT-PCR after stimulation with TNF (50 ng/ml), IL-1β (2ng/ml), PDGF (10ng/ml), TGFβ (20ng/ml), IL-17A (50ng/ml), IFNγ (10ng/ml), or IL-6 (20ng/ml) for 6 h. The graph shows fold change value compared with RA and FLS cultured in medium alone (n = 5–7 each). (B) Dose-response and kinetics of CDH6 induction by TGFß. Left side: mRNA expression of CDH6 in RA FLS stimulated with TGFβ (0, 0.02, 0.2, 2, or 20 ng/ml for 6 h) (n = 6). Right side: Time course of mRNA expression of CDH6 in RA FLS stimulated with 20 ng/ml TGFβ (0, 3, 6, 12, or 24 h) (n = 6). (C) CDH6 protein expression in RA FLS stimulated with TGFß (20 ng/ml for 24 h) (n = 4). All data were presented as mean ± SEM. Analysis was performed using the Friedman test (A and B) and Wilcoxon test (C). * p ≤ 0.05

Fig 5: Epigenetic profile of the CDH6 promoter region in RA FLS and OA FLS. (A) Genome browser view of open chromatin, H3K27ac modifications in the CDH6 promotor region. (B) Pileup visualization of open chromatin (OA: n = 11, RA: n = 11) calculated by area under curves (AUC). (C) RA and OA FLS were incubated with 1µM of ITF2357 (pan HDAC inhibitor) for 12 h and 24 h, and CDH6 mRNA levels were measured by qRT-PCR (n = 5). CDH6 was reduced after ITF2357 treatment in RA FLS (OA: n = 5, RA: n = 5). (D) Absolute counts of DNA methylation in CDH6 promoter region in RA FLS and OA FLS, no significant differences were found. All data were presented as mean ± SEM. * p ≤ 0.05 by unpaired t-test (B and D) and Kurskal-Wallis test (C)