Fluorescence-activated cell sorting (FACS) and clonogenicity analyses.

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Fluorescence-activated cell sorting (FACS) and clonogenicity analyses. Fluorescence-activated cell sorting (FACS) and clonogenicity analyses. A, effects of JMJD2B knockdown on cell-cycle kinetics in cancer cells. A549 cells were collected 72 hours after treatment with siRNAs, and cell-cycle distribution was analyzed by flow cytometry after coupled staining with FITC-conjugated anti-BrdU and 7-AAD, described in Materials and Methods. Representative histograms of the FACS results (top) and numerical analysis (bottom) are shown for each experiment. B, validation of subcellular localization and an enzymatic activity of JMJD2B. 3xFLAG-JMJD2B transfected HeLa cells were stained for specific antibodies as indicated above. Immunostaining showed nuclear localization of JMJD2B and its reversing activity against tri- and di-methylated H3K9, accompanied with the increased signal of H3K9me and the decreased signal of Hp1beta. Scale bar denotes 10μm. DAPI, 4′, 6-diamidino-2-phenylindole. C, clonogenicity assays of NIH3T3 cells. Cells transfected with a 3xFLAG-JMJD2B vector and a 3xFLAG-JMJD2B ΔJmjC were cultured in DMEM 10% FBS containing 0.9 (mg/mL) geneticin/G-418 for 2 weeks and colonies were stained with Giemsa. Gouji Toyokawa et al. Cancer Prev Res 2011;4:2051-2061 ©2011 by American Association for Cancer Research