Fig 2: The poly(I:C)- or LPS-induced association of c-Jun with the Ifnb promoter is prevented by BET inhibitors without inhibiting the phosphorylation of c-Jun or ATF2(A and B) The experiment was performed as in Figure 4 except that an anti-c-Jun antibody was used. (C and D) RAW cells were incubated for 1 h with 1.0 µM BI-2536, 1.0 µM JQ1 or 10 µM of the JNK inhibitor JNK-IN-8 and then stimulated for 1 h with 10 µg/ml poly(I:C) (C) or for 30 min with 100 ng/ml LPS (D). The cells were lysed and 20 µg of cell extract protein was denatured in SDS and subjected to SDS/PAGE followed by immunoblotting with antibodies that recognize the phosphorylated forms of JNK1/2, c-Jun and ATF2 as well as GAPDH. JNK-IN-8 binds covalently to JNK1 and JNK2 leading to a small decrease in their electrophoretic mobilities. IP, immunoprecipitation.

Fig 3: Infection with MRSA triggered NF-?B and MAPK signaling pathways associated with generation of immune responses, inflammation, and articular damage. (A) Transcriptome profiles were analyzed in primary BMDMs infected for four hours with MRSA using RNA-sequencing. We compared the normalized dataset with non-infected data, determined genes that were significantly increased in MRSA infection, and then analyzed canonical pathways alongside upstream regulating factors. The downstream genes regulated by NF-?B and FOS were verified and presented in the heatmap. (B) Human synovial tissue was infected with GFP-labeled MRSA (4×106 CFU) for 24 hours and GFP expression was measured as a marker for ongoing infection (Scale bar: 100 µm). The expression of p-NF-?B, NF-?B, I?-Ba, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 in whole protein extract obtained from MRSA-infected human synovial tissue was measured; ß-actin was used as a loading control. (C) Human primary synovial cells were infected with GFP-labeled MRSA (4×106 CFU) for 2 hours, at which time intracellular GFP expression was quantified (Scale bar: 100 µm). The expression of p-NF-?B, NF-?B, I?-Ba, p-ERK, ERK, p-JNK, JNK, p-p38, and p38 in whole protein extract derived from MRSA-infected synovial cells at two and four hours post-infection was measured; ß-actin was used as a loading control. Error bars show means ± SD. Two-tailed unpaired t-test analysis was used to assess statistical significance when compared to the normal group (**p < 0.01).

Fig 4: Increased K63-linked polyubiquitin in injured porcine articular cartilage. Porcine metacarpophalangeal (MCP) joints were equilibrated at 37°C for 1 hour. A, Cartilage was dissected and either snap-frozen (time point 0 minutes) or cultured for the indicated times. K63-linked ubiquitin (UB-K63) proteins were obtained from cartilage lysates using a pull-down assay with receptor associated protein 80 (RAP-80) ubiquitin interacting motifs (UIMs) as described in Materials and Methods, and proteins bound to the beads were analyzed by Western blotting (WB) with an anti–K-63–linked ubiquitin antibody. Lysates were analyzed by Western blotting for phospho-JNK (ph-JNK) and total ERK (T-ERK). Representative results from 3 independent experiments are shown. B, Porcine MCP cartilage sections (5–7 µm) were deparaffinized, rehydrated, and treated for 10 minutes with proteinase K for antigen retrieval. Endogenous peroxidase activity was blocked with 3% H2O2 for 10 minutes, followed by blocking with avidin–biotin for another 10 minutes. Sections were then blocked for 2 hours at room temperature with 10% goat serum in phosphate buffered saline, and incubated overnight at 4°C with anti-K63 ubiquitin antibody (1:50 dilution). Sections were washed and then incubated for 2 hours in the dark with secondary antibodies conjugated with streptavidin. Antibody binding was visualized using a Vectastain kit and DAB Peroxidase Substrate Kit (Vector). Slides were then counterstained with hematoxylin and mounted with DPX. Original magnification × 10. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39965/abstract.

Fig 5: Cartilage injury leads to activation of transforming growth factor ß–activated kinase 1 (TAK-1), and inhibition of TAK-1 abolishes injury-dependent inflammatory gene expression. A, Porcine metacarpophalangeal (MCP) joints were equilibrated at 37°C for 1 hour. Articular cartilage was dissected and either snap-frozen or kept in serum-free medium for the indicated times. Cartilage was then washed with 1× phosphate buffered saline and lysed in radioimmunoprecipitation assay buffer. Lysates were analyzed by Western blotting (WB) for phospho–TAK-1 (ph-T187), phospho-JNK, and ERK. B, A TAK-1 activity assay was carried out as described in Materials and Methods to confirm TAK-1 activation. IP = immunoprecipitation; ph-MBP = phospho–myelin basic protein. C and D, Porcine MCP joints were injected with 5 µM TAK-1 inhibitor 5z-7-oxozeanol (OXO) or DMSO (vehicle [V]), as described in Materials and Methods, and kept at 37°C for 1 hour. Joints were then opened and cartilage was dissected and snap-frozen (time point 0 minutes) or cultured for the indicated times with or without TAK-1 inhibitor. Cartilage lysates were analyzed for phospho-JNK and total ERK (T-ERK) (C) and for I?B and total ERK (D). E, Cartilage was snap-frozen (time point 0 hours) or kept at 37°C for 4 hours with or without 5 µM TAK-1 inhibitor (OXO). RNA was extracted from the tissue and used in quantitative reverse transcriptase–polymerase chain reaction studies of a panel of typical inflammatory response genes. Bars show the mean ± SEM of 3 independent experiments. * = P < 0.05; ** = P < 0.01, by unpaired t-test. NS = not significant; COX-2 = cyclooxygenase 2; IL-6 = interleukin-6; MMP-3 = matrix metalloproteinase 3; iNOS = inducible nitric oxide synthase; TNF = tumor necrosis factor. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/journal/doi/10.1002/art.39965/abstract.