Fig. 4
Expression of OPN and evaluation of TLR9-MYD88 signaling pathway in CD19 + cells from autoimmune mice. A. IHC for OPN performed on splenic tissues from BALB/c and autoimmune mice with and without lymphomas. Scale bar: 100 μm (left) and 50 μm (right). B. Representative double IF for OPN (red) and the endosomal marker CD63 (blu) on purified CD19 + cells from BALB/c and Faslpr/lpr mice. Scale bar: 15 μm. C. Flow cytometry analysis showing CD86 expression on CD19 + cells from naïve and autoimmune mice (n = 3) with or without 3-day stimulation with CpG 1826 (*, p < 0.05, **, p < 0.01, ***, p < 0.001, ****, p < 0.0001, Two-way ANOVA). D. Representative western blot showing the level of MYD88, IRAK4 and IRAK1 in CD19 + B cells purified from Faslpr/lpr and OPN-/-Faslpr/lpr mice at steady state condition and after TLR9-triggering with CpG 1826. The TLR4 agonist LPS and the TLR3 ligand Poly I:C were used as positive and negative controls, respectively. E. Western blot quantification relative to β-ACTIN housekeeping gene of MYD88, IRAK4 and IRAK1. F. Flow cytometry analysis showing the percentage of CD19 + TLR9 + cells (upper panel) and TLR9 protein level (MFI) on CD19 + cells (lower panel) from the spleen of Faslpr/lpr (n = 6) and OPN-/-Fas.lpr/lpr mice (n = 6). B/c and OPN-/-mice were used as controls. Data in the graphs are referred to a pool of two independent experiments. (**, p < 0.01; Ordinary one way ANOVA) (*, p < 0.05; Ordinary one way ANOVA)