ChIP

For the ChIP experiments, 1×10⁷ U-87MG ATCC cells were transfected with the JMJD3 vectors for 48 h at 37°C. Cross-linking with 1% formaldehyde for 10 min at room temperature, collection, sonication (non-contact sonication: time on 30 sec, time off 30 sec, 15 cycle at 4°C) and immunoprecipitation were performed using an EZ-ChIP™ kit (cat. no. 17-371; EMD Millipore) according to the manufacturer's protocol. Antibodies against H3K27me3 (cat. no. ab6002; 1:50; Abcam) were used for immunoprecipitation. Antibodies against RNA polymerase (cat. no. 17-371; 1:50; EMD Millipore) and normal mouse IgG (cat. no. 17-371; 1:50; EMD Millipore) were used as the positive and negative controls, respectively. ChIP-PCR was performed using a 2×Es Taq MasterMix (Dye) kit (CoWin Biosciences, Inc.) to detect the immunoprecipitated DNA fragments of the CXCL12 promoter. The thermocycling conditions were as follows: 94°C For 2 min, followed by 30 cycles of 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec, 72°C for 2 min. A 1% agarose gel and ethidium bromide were used for agarose gel electrophoresis. The primer sequences used for ChIP-PCR are listed in Table I.