Figure 2 : MANF was specifically induced in the FLS of AIA rabbit synovial tissue and relocalized to the nuclei under ER stress.

From: Mesencephalic Astrocyte-Derived Neurotrophic Factor Is Involved in Inflammation by Negatively Regulating the NF-κB Pathway

Figure 2

(a) Double-labeled immunofluorescence of CD68 (green) and MANF (red) in the synovial tissues of AIA rabbits. (b) Double-labeled immunofluorescence of a-SMA (green) and MANF (red) in the synovial tissues of AIA rabbits. CD68 and a-SMA were used as the markers of the macrophage-like synoviocytes (MLS) and the fibroblast-like synoviocytes (FLS), respectively. Scale bar = 50 µm. (c) MANF localization in the nuclei in some FLS in the inflammatory synovial tissue. The synovium of AIA rabbits was stained with anti-MANF (green) antibody. DAPI was used to stain the nuclei (blue). Scale bar = 50 µm. (d) MANF was relocalized to FLS nuclei after treatment with LPS and tunicamycin (TM). The FLS isolated from the synovium of normal rabbits were treated with LPS (10 μg/ml) for 48 hrs or TM (2.5 μg/ml) for 16 hrs. Then, the cells were stained with anti-MANF antibody (red) and DAPI (blue). Scale bar = 10 µm. The experiments in panel a-d were repeated for at least three times. (e) TM-induced nuclear localization of MANF in FLS was detected by Western blotting. The FLS isolated from AIA rabbits were treated with TM (2.5 μg/ml) for 16 hrs. The cytosolic and nuclear fractions were subjected to Western blotting with anti-MANF antibody. GAPDH and nucleolin were used as the markers of cytosol and nucleus, respectively. The values are expressed as the mean ± SD of three individuals. *** P<0.0001, compared with DMSO control. (f) LPS-induced MANF nuclear localization in FLS was detected by Western blotting. The FLS were treated with LPS (10 μg/ml) for 48 hrs. Western blotting was used to detect MANF expression in the cytoplasm and nucleus. Tubulin and histone H3 were used as the markers of cytosol and nucleus, respectively. The values are expressed as the mean ± SD of three individuals. ** P = 0.001, compared with PBS control.