Figure 3.

TIPE2 overexpression enhanced DR5 and caspase expression and downregulated the activation of NF-κB.

Notes: (A) The FLSs were stimulated with 0.1 mg/mL ZF1 in ZF1 groups or PBS in no ZF1 groups for 6 hours. The total protein was extracted, and the expressions of apoptosis-related and intrinsic mitochondrial pathway-related proteins and pNF-κB-p105/p50 proteins were analyzed by Western blot assay. (B) Flow cytometry detection of apoptosis with annexin V/PI in AA-FLSs stimulated with ZF1. Before stimulation with 0.04 mg/mL ZF1, the TIPE2^+/+-FLS group was pretreated with caspase inhibitor Z-VAD-FMK and the MIGR1-FLS group was pretreated with the NF-κB inhibitor Bay. (C) The FLSs were stimulated with 0.1 mg/mL ZF1 in ZF1 groups or without ZF1 in PBS groups for 6 hours. The total protein was extracted and the expressions of TIPE2 and DR5 proteins were analyzed using Western blot assay. (D) TIPE2 upregulated the expression of DR5 on FLSs. Flow cytometry detection of DR5 expression with anti-DR5 and mouse IgG-APC in AA-FLSs stimulated with 0.1 mg/mL ZF1 or without ZF1 for 12 hours was carried out. (E) TIPE2 up-regulated the expression of DR5 on FLSs. The data are presented as the mean ± SD of three independent experiments, ^***P<0.001. Data are representative of three independent experiments, which showed similar results.

Abbreviations: AA, adjuvant arthritis; DR, death receptor; FLS, fibroblast-like synoviocyte; NF-κB, nuclear factor-kappa B; PBS, phosphate-buffered saline; PI, propidium iodide; SD, standard deviation; APC, Allophycocyanin.