Synoviocyte preparation and treatment
This study was approved by the Institutional Review Board of Severance Hospital (4-2012-0733). Informed consent was obtained from RA patients providing synovial tissues. FLS and MH7A cells were cultured as described previously [11, 12]. FLS and MH7A cells were treated with synthetic ThiaMet-G (200 μM), an OGA inhibitor, for 6 h, followed by TNF-α (10 μg/mL) for 12 h [13]. We synthesized ThiaMet-G according to a method for its synthesis provided by Prof. Vodadlo DJ (Simon Fraser University, Canada). The proliferation/viability of FLS and MH7A was measured by MTT assay.
Overexpression and small hairpin RNA (shRNA)-mediated knockdown of OGA
The human OGA gene was cloned into a pRK5-FLAG vector (Genentech Inc., USA) and transiently transfected into MH7A cells using Lipofectamine 2000 (Invitrogen, USA). The pLKO.1 vector and packaging plasmid pMD.2G-VSVG and PsPax2 were used to generate a lentivirus-based OGA shRNA construct, as described previously [14]. This protocol is available on the website [15].
RT-PCR
The levels of mRNAs encoding pro-inflammatory molecules were quantitated by RT-PCR. All experiments were performed in triplicate using a 7300 RT-PCR System (Applied Biosystems, USA) and a SYBR Green kit (Takara, Japan). A full list of primers is available in Additional file 1.
Western blotting and succinylated wheat germ agglutinin (sWGA)-affinity purification
Western blotting immunoprecipitation was performed using anti-p65 (Santa Cruz Biotechnology, USA) and anti-O-GlcNAc (Covance, USA) and succinylated wheat germ agglutinin (sWGA)-affinity purification were performed for precipitating the O-GlcNAcylated proteins [16].
Measurement of DNA binding affinity and transcriptional activity of p65
Avidin-biotin complex DNA (ABCD) lysis buffer containing 10 mM Tric-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA and 0.2 % Nonidet P-40 with protease inhibitor cocktail was used for MH7A cells lysis to measure DNA binding affinity. Lysates were incubated with biotinylated NF-κB promoter site DNA and streptavidin beads, washed three times in ABCD lysis buffer, and eluted in SDS sample buffer. NF-κB transcriptional activity was measured in MH7A cells transfected with NF-κB-dependent luciferase reporter gene construct 4xkB- Luc. Reporter gene assays were performed using the Dual-Luciferase kit (Promega, USA), with firefly luciferase activity normalized to renillia luciferase activity.
Mice with CIA
We evenly divided 30 male DBA/1 mice at 8 weeks of age (SLC, Shizoka, Japan) into three groups (group 1, controls; group 2, untreated; group 3, ThiaMet-G-treated). We established a CIA model and assessed its severity clinically (arthritis score and paw thickness) and histologically (synovial proliferation, bone destruction and inflammatory cell infiltration under hematoxylin and eosin stain), as described previously [17]. Two weeks after primary immunization, mice were treated with 20 mg/kg ThiaMet-G via intraperitoneal (IP) injection, every other day for 26 days [18]. Controls and untreated mice were injected IP with the same volume of PBS every other day during the ThiaMet-G treatment period. Mice were next sacrificed and immune cells isolated from the spleen; T cells were then isolated and analyzed using fluorescence-activated cell sorting (FACS) [19]. The experimental protocols were approved by the Institutional Animal Care and Use Committee of Yonsei University College of Medicine.
Statistical analyses
Each experiment was performed in triplicate, and repeated three to four times. All of the analyses were conducted using the SPSS package for Windows version 20.0 (SPSS Inc., Chicago, IL, USA). Data are representative of the mean values obtained. Differences between groups were evaluated using the Mann–Whitney U test. Multiple-group comparisons were performed using analysis of variance followed by application of Tukey’s method for multiple comparisons. P values <0.05 were considered to indicate statistical significance for all statistical evaluations.
