Fig 2: CDH6 regulates RA FLS cell survival. (A) Effect of CDH6 knockdown on FLS growth. OA and RA FLS were transfected with siCDH6 or CT and cultured in the presence of PDGF (10 ng/mL) for 4 and 7 days in triplicate wells. Data shows the percentage of inhibition after knockdown CDH6 in cell growth in RA FLS (n = 10) and OA FLS (n = 7), RA FLS is significantly higher than OA FLS. Graphs represent the percentage of inhibition of cell growth induced by siCDH6 compared to CT in both RA and OA FLS. (B) Cell cycle analysis after CDH6 knockdown and stimulated by PDGF. RA FLS were transfected with siRNA, serum-starved, and induced with 10ng/ml of PDGF. Representative histograms of DNA content in RA FLS lines transfected with CT and siCDH6 in 0 h and 24 h (n = 3). CDH6 had no effect on any phase of the cell cycle. (C) Autophagy analysis after CDH6 knockdown. FLS were cultured with PDGF (10 ng/mL) for 15, 30, 60, and 180 min with or without knockdown. The autophagy marker LC3 and GAPDH protein were measured by Western blot analysis. Representative images of LC3 cultured with or without PDGF in CT and siCDH6 transfected cells are shown (n = 4). (D) Effect of CDH6 knockdown on FLS apoptosis. FLS transfected with siCDH6 and cultured with or without PDGF (10 ng/ml), then incubated for 15 h using the Apo-ONE® Homogeneous Caspase-3/7 Assay kit (n = 6). Data is shown as the percentage of the ratio between siCDH6 and the control, showing apoptosis is higher after knockdown. All data were presented as mean ± SEM. *P ≤ 0.05, Kruskal-Wallis multiple comparison test (A) and ANOVA multiple comparison test (C and D)

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: 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 5: 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