Fig 1: Therapeutic effects of AS1411‐OSMI alone or in combination with ETA in vivo. (A) Experimental design for CIA mouse models. DBA/1J mice were immunized with CII emulsified in CFA to induce CIA. Following CIA establishment, CIA mice received systemic treatments with Veh, AS1411, OSMI‐1, AS1411/OSMI Mix, AS1411‐OSMI, ETA, or ETA + AS1411‐OSMI for 25 days. The dosage of AS1411OSMI was determined as 2.5 µmol/kg based on preliminary doseresponse experiments, in which this concentration yielded maximal OGT degradation in vivo. AS1411, OSMI‐1, or AS1411/OSMI Mix were given at equimolar doses for direct comparison. ETA was intraperitoneally administered at 5 mg/kg, and non‐immunized mice served as healthy controls. (B) Arthritis scores of CIA mice from different treatment groups. n = 5. (C) Micro‐CT images of the hind paws of healthy controls or CIA mice from different treatment groups. n = 5. (D) Representative images of SO/FG and HE staining on sections of the hind paws of healthy controls or CIA mice from different treatment groups. n = 5. Scale bars, 200 µm. (E) Quantification of synovitis, bone, and cartilage erosion on SO/FG‐ and HE‐stained sections. n = 5. (F) IF staining of OGT, SAP130, BTG2, and CXCL12 on sections of the hind paws of healthy controls or CIA mice from different treatment groups. n = 5. Scale bar, 100 µm. (G) Quantification of positive cells (%) for IF staining of OGT, SAP130, BTG2, and CXCL12. n = 5. Data are presented as mean ± SD, and statistical significance is calculated by two‐way ANOVA (B) and one‐way ANOVA with Tukey's test (E and G).
Fig 2: Stabilization of SAP130 by OGT‐mediated O‐GlcNAcylation. (A) Workflow of LC‐MS/MS‐based analysis for identifying OGT‐interacting proteins and their potential O‐GlcNAcylation sites. Briefly, RA‐FLSs were lysed, followed by protein digestion, peptide separation, and LC‐MS/MS detection. The right panel summarizes the identified protein candidates and their potential O‐GlcNAcylation modification sites obtained via this workflow. (B and C) Co‐IP assays for the interaction between OGT and SAP130 in RA‐FLSs using an anti‐OGT antibody (B) and an anti‐SAP130 antibody (C), respectively. n = 3. (D) Co‐IP assay for the interaction between OGT and SAP130 in 293T cells overexpressing HA‐tagged SAP130 (HA‐SAP130), after transfection with the Flag‐empty vector or the vector expressing Flag‐tagged OGT (Flag‐OGT). n = 3. (E and F) Co‐IP assays for the interaction between OGT and SAP130 in vitro using an anti‐OGT antibody (E) and an anti‐SAP130 antibody (F), respectively. 2 µg/ml Flag‐tagged rhOGT was incubated with 2 µg/ml Myc‐tagged rhSAP130 for 7 h before the co‐IP assay. 15% of the rhOGT‐rhSAP130 mixture was used as input. n = 3. (G) WGA pull‐down assay for determining O‐GlcNAcylation levels of SAP130 in RA‐FLSs. n = 3. (H) In vitro O‐GlcNAcylation assays with Myc‐SAP130 as a substrate, in the presence of UDP‐GlcNAc, OGT, or OGA. n = 3. (I) O‐GlcNAcylation levels of SAP130 in RA‐FLSs transfected with the Flag‐empty vector or the vector expressing Flag‐OGT, in the presence of 5 µM proteasome inhibitor MG132. n = 3. (J) O‐GlcNAcylation levels of SAP130 in RA‐FLSs transfected with siCtrl, siOGT‐1, or siOGT‐2, in the presence of 5 µM MG132. n = 3. (K) Potential O‐GlcNAcylation modification sites (T281, S282, T296, S316, and T473) on SAP130 identified via LC‐MS/MS analysis. (L) Co‐IP assay using anti‐Myc antibody beads to assess O‐GlcNAcylation levels of overexpressed Myc‐SAP130 WT and its mutants (T281A, S282A, T296A, S316G, or T473A) in 293T cells. n = 3. (M and N) mRNA (M) and protein (N) levels of SAP130 in RA‐FLSs transfected with siCtrl, siOGT‐1, or siOGT‐2. n = 3. (O) SAP130 protein levels in RA‐FLSs transfected with siCtrl, siOGT‐1, or siOGT‐2, in the presence of 5 µM MG132 or 400 nM BafA1. n = 3. (P) Ubiquitination levels of SAP130 in RA‐FLSs transfected with siCtrl, siOGT‐1, or siOGT‐2, in the presence of 5 µM MG132. n = 3. (Q) Ubiquitination levels of overexpressed Myc‐SAP130 WT and its mutants (T281A, S282A, T296A, S316G, or T473A) in 293T cells. n = 3. (R) Schematic model illustrating that OGT‐mediated O‐GlcNAcylation stabilizes SAP130 by inhibiting its ubiquitination and proteasomal degradation. Data are presented as mean ± SD, and statistical significance is calculated by one‐way ANOVA with Dunnett's test (M).
Fig 3: Epigenetic regulation of BTG2 by the OGT‐SAP130 axis. (A) Volcano plot of differentially expressed genes in RA‐FLSs transfected with siOGT versus siCtrl, as determined by RNA sequencing. n = 3. Fold change > 2, P < 0.05. (B) Volcano plot of differentially expressed genes in RA‐FLSs transfected with siSAP130 versus siCtrl, as determined by RNA sequencing. n = 4. Fold change > 2, P < 0.05. (C) Venn diagram showing the overlapping set of downregulated genes in RA‐FLSs transfected with siOGT (versus siCtrl, n = 3) and those from siSAP130 (versus siCtrl, n = 4). (D) Pathway enrichment analysis of the overlapping downregulated genes in RA‐FLSs transfected with siOGT (versus siCtrl, n = 3) and those from siSAP130 (versus siCtrl, n = 4). Fold change > 2, P < 0.05. (E) Levels of histone modifications (H3K9ac, H3K14ac, H3K27ac, and H3K27me3) in RA‐FLSs after transfection with siCtrl, siSAP130‐1, or siSAP130‐2. n = 3. (F) Colocalization of OGT with SAP130 in RA‐FLSs. n = 3. Scale bars, 50 µm. (G) Venn diagram showing the overlapping set of upregulated genes in RA‐FLSs transfected with siOGT (versus siCtrl, n = 3) and those from siSAP130 (versus siCtrl, n = 4). (H) Pathway enrichment analysis of the overlapping upregulated genes in RA‐FLSs transfected with siOGT (versus siCtrl, n = 3) and those from siSAP130 (versus siCtrl, n = 4). Fold change > 2, P < 0.05. (I) Heatmap of the top 30 upregulated genes in RA‐FLSs transfected with siOGT versus siCtrl. n = 3. (J) Heatmap of the top 30 upregulated genes in RA‐FLSs transfected with siSAP130 versus siCtrl. n = 4. (K and L) mRNA (K) and protein (L) levels of BTG2 in RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. n = 3. (M) ChIP‐qPCR analysis of HDAC1, H3K27me3, and H3K27ac at the BTG2 promoter in RA‐FLSs transfected with siCtrl or siSAP130. n = 3. (N) BTG2 protein levels in RA‐FLSs transfected with siCtrl, siOGT‐1, or siOGT‐2. n = 3. (O) BTG2 protein levels in SAP130‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector. n = 3. (P) BTG2 protein levels in HDAC1‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector. n = 3. (Q) The regulatory model of the OGT‐SAP130‐BTG2 axis. Data are presented as mean ± SD, and statistical significance is calculated by one‐way ANOVA with Dunnett's test (K) and unpaired t‐test (M).
Fig 4: SAP130‐dependent aggressive transformation of RA‐FLSs driven by OGT. (A) IF staining of synovial tissues from healthy controls and RA patients using anti‐SAP130 and anti‐THY1 antibodies. n = 3. Scale bars, 25 µm. (B) IF staining of synovial tissues from healthy controls and CIA mice using anti‐SAP130 and anti‐THY1 antibodies. n = 5. Scale bars, 25 µm. (C) SAP130 mRNA levels in synovial tissues from OA and RA patients. n = 10. (D and E) mRNA (D) and protein (E) levels of SAP130 in RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. n = 3. (F) Apoptosis of RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2, as determined by flow cytometry. Quantification is shown on the right. n = 3. (G) Proliferation of RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2, as measured by EdU staining. n = 3. Scale bars = 100 µm. (H) Colony formation of RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. Quantification is shown on the right. n = 3. (I) Transwell invasion of RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. Quantification is shown on the right. n = 3. Scale bars, 50 µm. (J) Transwell migration of RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. Quantification is shown on the right. n = 3. Scale bars, 50 µm. (K) mRNA expression of CXCL5, CXCL12, JAK2, and JAK3 in RA‐FLSs transfected with siCtrl, siSAP130‐1, or siSAP130‐2. n = 3. (L) Apoptosis of SAP130‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector, as measured by TUNEL staining. n = 3. Scale bars, 100 µm. (M) Proliferation of SAP130‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector, as measured by EdU staining. n = 3. Scale bars, 100 µm. (N) Transwell invasion of SAP130‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector. Quantification is shown on the right. n = 3. Scale bars, 50 µm. (O) Transwell migration of SAP130‐silenced RA‐FLSs after transfection with the empty vector or the OGT‐overexpressing vector. Quantification is shown on the right. n = 3. Scale bars, 50 µm. Data are presented as mean ± SD, and statistical significance is calculated by the Mann‐Whitney U test (C), one‐way ANOVA with Dunnett's test (D, F, and H‐K), and Tukey's test (N and O).
Supplier Page from Abcam for Anti-SAP130 antibody