Fig. 3: Reactivity of E4 to mouse/human joint tissues.

A Immunofluorescence (IF) staining of E4/L2/E4m on naïve or CAIA DBA/1 mouse joint tissues taken on day 15 of CAIA. Antibodies were stained with goat-anti-mouse IgG antibody conjugated with CF488A and DNA was stained with Hoechst. Images were captured by confocal microscopy at ×10 magnification and the scale bars represent 200 μm. B IF staining of E4/L2/E4/ACC4 on naïve or arthritic joint tissue from FCGR2B KO mice taken on day 1 of CAIA. The staining was performed as above. Images were captured by confocal microscopy at ×10 magnification and the scale bars represent 200 μm. C IF staining of E4/L2/E4m on citrullinated extracellular matrix (ECM) from ATDC5 chondrocytes. Cells were cultured for 14 days with insulin-transferrin-selenium (ITS) following a treatment with recombinant human PAD4 (10 μg/ml) or PBS overnight before staining. E4/L2/E4m was stained as above, the citrullinated CII was labeled using biotinylated ACC4 antibody and stained with streptavidin-PE, and DNA was stained with Hoechst33342. Images were captured by confocal microscopy at 20× magnification. The scale bars represent 100 μm; D antibody-bound areas (n = 3 independent samples) were analyzed using ImageJ software. Data were assessed using one-way ANOVA and presented as mean ± SD. E IF staining of E4/L2/E4m on human cartilage explants from RA patients (RA01 & RA02) and a non-RA donor (HC01). Antibodies were stained as above. Images were captured by confocal microscopy at 10× magnification. The scale bars represent 200 μm.