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. 2022 Oct 14;12:17242. doi: 10.1038/s41598-022-22230-z

Figure 2.

Figure 2

Increased HYBID expression by IL-6 in OA chondrocytes. (a) and (b) Effects of IL-6 on the mRNA and protein expression of HYBID in OA chondrocytes. OA chondrocytes at P2 were treated with IL-6 (0, 10, 50 or 100 ng/ml) and soluble IL-6 receptor (sIL-6R; 100 ng/ml). Cell lysates were harvested at 24 h after cultures for the HYBID mRNA expression and at 48 h for protein expression and subjected to quantitative real-time PCR using the ΔΔCt method (a) and immunoblotting with anti-HYBID antibody, followed by densitometric analysis (b). The average HYBID:GAPDH ratio in control OA chondrocytes treated with vehicle alone was set at 1. (c) Demonstration of IL-6-mediated HYBID protein overexpression in OA chondrocytes. OA chondrocytes at P2 were stimulated with IL-6 (0 or 100 ng/ml) and sIL-6R (100 ng/ml) in the presence of non-immune IgG (NI-IgG, 25 µg/ml) or anti-IL-6R antibody (tocilizumab, 25 µg/ml) for 48 h, and cell lysates were subjected to immunoblotting with anti-HYBID antibody, followed by densitometric analysis. (d) and (e) Effects of IL-6 on the mRNA and protein expression of TMEM2 in OA chondrocytes. OA chondrocytes at P2 were treated with IL-6 (0, 10, 50 or 100 ng/ml) and sIL-6R (100 ng/ml) for 24 h and 48 h, and TMEM2 expression was measured by quantitative real-time PCR using the ΔΔCt method (d) and immunoblotting, which was followed by densitometric analysis (e). The average TMEM2:GAPDH ratio in control OA chondrocytes treated with vehicle alone was set at 1. Values are expressed mean ± SD (n = 3). *, P < 0.05. The uncropped full-length gels can be found in Supplementary Figure S3.