Figure 1.
Clinical arthritis symptoms in collagen-injected TXNDC5-Tg mice.
The incidence of arthritis (A), the hind paw clinical score (B) and the hind paw thickness (C) in TXNDC5-Tg (filled squares, n = 15) and WT (filled circles, n = 10) mice were measured at various time points following the first injection. A separate group of TXNDC5-Tg mice received BSA, and these mice were used as normal controls (empty squares, n = 10). (D) The symptoms of CIA were observed in the hind paws of these collagen-treated mice. a) represents collagen-injected TXNDC5-Tg, b) represents BSA-treated TXNDC5-Tg, c) represents collagen-injected wild type. * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 2.
Histochemical examination of collagen-induced arthritis in experimental mice.
The tissue structures of the paw (A–C), knee (E–F) and ankle (G–I) were histochemically examined. Synovial hyperplasia and inflammation, cartilage destruction and bone resorption with pannus formation were observed in the arthritic joints of the collagen-treated TXNDC5-Tg mice (n = 5) but not in WT (n = 5) and TXNDC5-Tg mice treated with BSA (n = 5). Single arrow represents synovial membrane, and double arrow indicates joint cartilage. Magnification: 200×.
Figure 3.
Measurement of cytokine levels using ELISA.
(A) IL-1α, IL-1β, TNF-α and IL-17 levels in the sera of TXNDC5-Tg (n = 5) and WT mice following the injection of bovine collagen II (n = 5) and TXNDC5-Tg mice injected with BSA (n = 5). TXNDC5-Tg mice exhibited higher sera levels of TNF-α, IL-1α, IL-1β and IL-17 than wild type mice with treatment. (B) IL-1α, IL-1β, TNF-α and IL-17 levels in the supernatant of TXNDC5 siRNA (100 nM)-treated RASFs (n = 15). Parallel experiments with Mm/Hs-MAPK1 siRNA and Allstar siRNA were used as controls. RASFs without siRNA were also used as controls. TNF-α, IL-1α, IL-1β and IL-17 levels decreased significantly in RASF supernatants following treatment with TXNDC5 siRNA compared to the normal controls without the siRNA treatment. * = p<0.05, ** p<0.01, *** = p<0.001.
Figure 4.
TXNDC5 and adiponectin expression in TXNDC5 siRNA-treated RASFs.
The RASFs were transiently transfected with TXNDC5 siRNA (n = 15), and the effects of TXNDC5 knockdown were determined at the mRNA (A) and protein levels (B) using real-time PCR and Western blotting, respectively. The effect of inhibiting TXNDC5 expression on adiponectin expression was also determined using real-time PCR (C) and Western blotting (D). The mRNA levels of TXNDC5 and adiponectin were normalized to β-actin. The protein levels of TXNDC5 and adiponectin were normalized to GAPDH. TXNDC5 and adiponectin levels were not significantly changed between the positive control, the negative control and the cells without siRNA treatment. PC indicates the positive control, and NC indicates the negative control. * = p<0.05, ** p<0.01, *** = p<0.001.
Figure 5.
TXNDC5 expression in RASFs (n = 15) following treatment with different concentrations of CoCl2.
(A) HIF-1α expression was detected in RASFs prior to and following 1 µM CoCl2 treatment. Real-time PCR and Western blotting analyses were used to measure TXNDC5 mRNA (B) and protein expression (C). * = p<0.05, ** p<0.01, *** = p<0.001.
Figure 6.
Cell proliferation and cell invasion in RASFs (n = 15) treated with CoCl2 and TXNDC5-siRNA.
(A) The cell viability of RASFs was determined using an MTT assay. (B) The invasive ability of RASFs was examined using a 2-compartment transwell system. The average number of cells that invaded through the filter was quantified. (C) Crystal violet staining of the lower surface filters revealed the cells that passed through the filter and attached to the lower side of the filter (100×). The data were obtained from three independent experiments. * = p<0.05, ** p<0.01, *** = p<0.001.