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

Figure 4. R-2HG-treated stromal cells enhance proliferation and chemoresistance of AML cells.

Figure 4

(a) 293 T cells were transfected with different IDH mutants and the conditioned medium was collected to treat StromaNKtert cells. Protein level of COX-2, p65 and VCAM-1 in StromaNKtert cells was investigated. (b) The R-2HG level in the conditioned medium of control and IDH2/R172M-expressing 293 T cells was determined by mass spectrometry. *p < 0.05. (c) The intracellular level of R-2HG in stromal cells treated with the conditioned medium was also determined. *p < 0.05. (d) StromaNKtert cells were treated without (control) or with 20 mM R-2HG for 48 h. Conditioned media were collected and were was used to treat KG-1a cells. After different times, cell viability was studied by MTT assay. *p < 0.05. (e) StromaNKtert cells were treated without (control) or with 20 mM R-2HG for 48 h. Conditioned medium of R-2HG-treated StromaNKtert cells was pre-incubated with control IgG or IL-6 neutralizing antibody and then used to treat KG-1a cells. Cell viability was studied by MTT assay.*p < 0.05. (f) KG-1a cells or IDH2/R712M-expressing KG-1a cells were treated with sunitinib (1.5 μM) and cellular viability was studied. *p < 0.05. (g) KG-1a cells were treated without or with sunitinib (1.5 μM) in the absence or presence of conditioned medium of R-2HG-incubated StromaNKtert cells. *p < 0.05. (h) KG-1a cells were treated with 20 mM R-2HG and the expression of VLA-4 was studied. *p < 0.05. (i) KG-1a cells pre-stained with PKH26 were mixed with StromaNKtert cells. The mixed cells were treated with R-2HG, sunitinib or anti-VCAM-1 antibody for 48 h. In the last lane, StromaNKtert cells were first transfected with p65 shRNA for 24 h and then co-cultured with KG-1a cells in the presence of R-2HG and sunitinib. Apoptotic KG-1a cells (PKH26 and Annexin V double-positive cells) and viable KG-1a cells (PKH26-positive and Annexin V-negative cells) were analyzed by flow cytometry. *p < 0.05. (j) Chemoresistance assay was performed as described in (i) except the cytotoxic drug used was cytarabine.