Figure 4

IL-17 reverses the suppression of gene expression imposed by RNA-binding protein AUF1. (a and d) WT MSCs and auf1^−/− MSCs (a) or shCTRL MSCs and shAUF1 MSCs (d) were treated with IFNγ and TNFα, or together with IL-17 (all cytokines supplemented at 10 ng/ml) for 12 h, and iNOS mRNA expression was measured by quantitative RT-PCR. (b) WT MSCs were treated with IFNγ and TNFα (10 ng/ml), or together with different concentrations of IL-17; auf1^−/− MSCs were treated with IFNγ and TNFα (10 ng/ml), or together with 10 ng/ml of IL-17. After 24 h, cells were harvested for detection of iNOS by western blotting analysis. (c) MSCs were stably transfected with plasmid expressing shRNA against AUF1 or plasmid encoding scrambled sequences (CTRL), and cells were collected for identification of AUF1 by quantitative RT-PCR and Western Blot. These cells are referred to as shAUF1 MSCs or shCTRL MSCs. (e) shCTRL MSCs or shAUF1 MSCs were stimulated with IFNγ and TNFα, or together with IL-17 (all cytokines supplemented at 10ng/ml) for 12 or 24 h. iNOS expression was detected by western blot. (f) WT MSCs or auf1^−/− MSCs were first treated with IFNγ and TNFα, with or without IL-17 (10 ng/ml), IFNγ and TNFα were supplemented at different concentrations, for 12 h, and then co-cultured with T-cell hybridoma A1.1 cells at a ratio of 1 : 10 for 12 h. T-cell proliferation was measured by ³H-thymidine incorporation, taking the proliferation level of A1.1 alone as 100%. mRNA expression values are means±S.E.M. of three wells from a representative of three independent experiments. Western blotting data are representative of three independent experiments. Proliferation values are means±S.E.M. of three wells from a representative of three independent experiments