Unknown

Synovial tissues were obtained from patients with active RA (6 females and 1 male, aged 45–67 years) who were undergoing synovectomy or joint replacement. RA FLS were cultured in serum-free DMEM/F12 for 24 h at a density of 1 Ч 104 cells/well in 96-well plates in triplicate. After starvation, the cells were untreated (as negative control) or treated with 0.1% dimethyl sulfoxide (DMSO) (#D2650, Sigma, USA) or various concentrations of PLM (Piperlongumine) (5, 10, 15, 20 μM) for 48 h in DMEM/F12 with 10% FBS. After this period, the cells were cultured in FBS-free DMEM/F12 for 12 h. Ten microliters 5% MTT (dissolved in PBS, PH 7.4, Sigma, USA) was added to the cells, which were incubated for 4–6 h. The cells were washed twice with PBS, followed by the addition of 100 μl DMSO. The cells were shaken gently for 10 min at 37 °C to achieve complete dissolution. The absorbances were read on a spectrophotometer at wavelengths of 570 and 620 nm.