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. 2016 Aug 31;6:32428. doi: 10.1038/srep32428

Figure 3. R-2HG stimulates NF-κB-dependent gene transcription in bone marrow stromal cells.

Figure 3

(a) StromaNKtert cells were treated without (control) or with 20 mM R-2HG for 48 h. Conditioned medium was analyzed by cytokine array and the cytokines upregulated by R-2HG were indicated by red square. Averaged results of two independent experiments were shown in the right panel. (b) Cells were treated with 20 mM R-2HG for different times and the expression of NF-κB target genes were studied. *p < 0.05. (c) Protein level of COX-2, p65, and VCAM-1 in R-2HG-treated cells was investigated by Western blotting. (d) StromaNKtert cells were transfected with control or p65 shRNA for 24 h and were treated without or with 20 mM R-2HG for another 48 h. The protein level of COX-2, p65, IL-6 and VCAM-1 were studied (left panel). The mRNA level of COX-2, IL-6, VCAM-1, IL-8, and C5 was investigated by real-time RT-PCR (right upper panel). **p < 0.01 and *p < 0.05. Cells were also treated with p65 inhibitor peptide for 24 h and then stimulated with 20 mM R-2HG for another 48 h. The mRNA level of various NF-κB target genes was investigated by real-time RT-PCR (right lower panel). **p < 0.01 and *p < 0.05. (e) Primary bone marrow stromal cells (purchased from Cambrex Corporate) were treated with 20 mM R-2HG and the expression of COX-2, IL-6 and VCAM-1 were studied. *p < 0.05. (f) Expression of cytokines and cell adhesion molecules in StromaNKtert, normal primary bone marrow stromal cells and IDH-mutated patient’s bone marrow stromal cells (B199, B59F and B204). *p < 0.05. (g) Cytokine array was used to determine the cytokines released by stromal cells of three IDH-mutated patients. *p < 0.05.