Fig. 2

IL-1β induces LPCAT3 expression in human and mice chondrocytes. A The human chondrocytes were treated with IL-1β (10ng/ml) for 6-24h, the cell medium was collected and the cells were lysed in 2 × SDS sample buffer. The expression of LPCAT3 in the cell lysate was analyzed by immunoblotting using GAPDH as a loading control. Densitometric analysis of immunoblots was performed and the data was presented on the right side. Data are shown as mean ± SD of 3 independent experiments. *p < 0.05; ***p < 0.001. B, C The total RNA was isolated from the IL-1β-stimulated chondrocytes (24 h) and expression of MMP3 (B) and ADAMTS5 (C) mRNA was analyzed by RT-PCR. D, E, F, G The IL-1β-induced (24 h) secretion of cytokines TNFα (D), IL-6 (E), and eicosanoids, PGE₂ (F) and LTB₄ (G) in the medium were analyzed by ELISA, ***p < 0.001. H The human chondrocytes were pretreated with vehicle, mepazine (10 μM), or SU3327 (10 μM) for 1h, and the cells were stimulated with IL-1β for 24 h. The cells were lysed in 2 × SDS sample buffer and expression of LPCAT3 in the cell lysate was analyzed by immunoblotting. Densitometric analysis of immunoblots was performed and the data was presented on the right side. Data are shown as mean ± SD of 4 independent experiments. ***p < 0.001; ns: not significant. I The human chondrocytes were transfected with 200 nM of scrambled RNA (scRNA) or LPCAT3 siRNA. 48 h post-transfection, the cells were lysed in 2 × SDS-sample buffer, the proteins were separated by SDS-PAGE and the efficiency of knockdown was analyzed by immunoblotting. The LPCAT3-specific siRNA transfection inhibited the expression of LPCAT3 by 85% in chondrocytes. J The cells were lysed at different time intervals as shown in the figure and total lipids were isolated. The fatty acid methyl esters (FAME) were analyzed by gas chromatography. The arachidonic acid content in the cells was presented as a percent of total fatty acids. Data are shown as mean ± SD of 3 independent experiments. Ns- not significant; *p < 0.05; **p < 0.01; ***p < 0.001. K, L, M The LPCAT3 silenced chondrocytes were treated with IL-1β for 24 h and the cell medium was collected. The eicosanoids, PGE₂ (K), LTB₄ (L), and TNFα (M) were analyzed in the medium by ELISA. Data are shown as mean ± SD of 4 independent experiments. ***p < 0.001. N The human cartilage explants were transfected with 200 nM of scrambled RNA (scRNA) or LPCAT3 siRNA. 48 h post-transfection, the cells were lysed in 2 × SDS-sample buffer, the proteins were separated by SDS-PAGE and the efficiency of knockdown was analyzed by immunoblotting. O, P, Q The LPCAT3 silenced human cartilage explants were treated with IL-1β for 24 h and the cell medium was collected. The sGAG in the medium was analyzed by DMMB assay (O), and The PGE₂ (P) and LTB₄ (Q) were analyzed in the medium by ELISA. R, S The human chondrocytes were transfected with 200 nM of scrambled RNA (scRNA) or LPCAT3 siRNA. 48 h post-transfection, cells were treated with IL-1β for 24 h, and cell viability (MTT assay) (R), and cell death were analyzed by ELISA (S). Data are shown as mean ± SD of 3 independent experiments. ***p < 0.001