Fig 1: Elevated α(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 α(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 α(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 α(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 α(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.
Fig 2: Expression of RANK (Tnfrsf11a), TLR2 (Tlr2), and tartrate-resistant acid phosphatase (TRAP) (Acp5) is elevated during the commitment of monocytes to a tartrate-resistant acid phosphatase-positive (TRAP+) lineage.(A) Dimensional reduction projection of monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups onto two dimensions using principal components, depicting values of Slingshot pseudotime followed by gene expression levels of St3gal4, Tlr2, Tnfrsf11a, and Acp5. (B) A heat map of dynamic expression of St3gal4, Tlr2, Tnfrsf11a, and Acp5 in monocyte and macrophage cells expressing RANK, TRAP, and/or TLR2 from all sample groups. Pseudotime values and annotated cell types are depicted in the color-coded bars atop the heat map. (C) Representative images of TRAP staining and immunofluorescence co-staining for RANK (red), TLR2 (green), and DAPI (blue) staining for nuclei for bone marrow macrophages cultured with macrophage colony stimulating factor (M-CSF) ( + ng/ml) and RANKL (200 ng/ml) for 0, 1, 3, and 5 days. Scale bar, 0.1 mm. (D) Quantitative analysis of the percentage of TRAP+ cells in bone marrow macrophages at different time points (n=4 or 5, one-way ANOVA with Tukey’s multiple comparisons test). (E–G) Quantitative analysis of the mean intensity of RANK and TLR2 positive fluorescence, and the percentage of RANK+TLR2+ positive cells in bone marrow macrophages at different time points (n=5 or 6, one-way ANOVA with Tukey’s multiple comparisons test). All data are means ± SD. N.S=No Significant difference, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Supplier Page from Abcam for Anti-RANK antibody [64C1385]