Fig 2: Elevated a(2,3) sialylation in RANK+TLR2+ monocytes is associated with an increase in the transcription of sialyltransferases in CIA mice.(A) Representative histograms of sialic acid a(2,3)-modified cells in the bone marrow of DBA control mice and CIA mice 2 months after the first immunization. (B) Quantitative analysis of the frequency of sialic acid a(2,3)-positive cells in DBA mice and CIA mice (n=4, t-test). (C) Representative images of the biotin fluorescence staining for sialic acid a(2,3) (green) and of DAPI staining (blue) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. (D) Quantitative analysis of the mean intensity of sialic acid a(2,3)-positive fluorescence of the images represented in (C) (n=6, t-test). (E) Heat maps of dynamic expression of marker genes Tlr2, Tnfrsf11a, Acp5, sialyltransferases, and Fos in the RANK+TLR2- myeloid cells (top) and RANK+TLR2+ myeloid cells (bottom). (F) Representative images of the immunofluorescence staining for c-Fos (green) of the knee joint section from DBA mice and CIA mice 2 months after the first immunization. Scale bar, 0.5 mm. (G) Quantitative analysis of the mean intensity of c-Fos-positive fluorescence in (F) (n=5, t-test). (H–I) The mRNA expression changes of c-Fos in the knee joint tissue of CIA and DBA mice (H) or in the RANK+TLR2- and RANK+TLR2+ monocytes (I) (n=3, t-test). (J) Representative images of the immunofluorescence co-staining for RANK (red) and ST3GAL4 (green) of the knee joint sections from DBA mice and CIA mice 2 months after the first immunization. (K) Quantitative analysis of the RANK+ST3GAL4+ cells in (J) (n=5, t-test). (L) The electrophoresis of CHIP assay for the binding sites of c-Fos in the promoter sequence of the St3gal4 gene. (M) The diagram of different locations and sequences of the putative binding sites for c-Fos in the promoter of St3gal4 gene. *p<0.05, ***p<0.001, ****p<0.0001. Figure 6—source data 1.Gel blot of CHIP assay of St3gal4 gene expression.