To further investigate the role of ODC in ESCC progression, we introduced shODC into two human ESCC cell lines, KYSE450 and KYSE510, by lentiviral vector and examined the effect of ODC on proliferation and anchorage-independent growth. To select the most effective shRNA, we screened 5 different shRNA sequences targeting ODC. Our data showed that, shODC #2 and #4 worked well in KYSE450 cells, whereas shODC#3 and #4 worked well in KYSE510 cells (Fig. 2a). These shODC sequences were used for experiments. Knocking down ODC expression reduced ODC enzyme activity in both ESCC cell lines by more than 70% (Fig. 2b). Correspondingly, cell proliferation and colony formation in soft agar were also suppressed (Fig. 2c, d), indicating that ODC promotes the progression of ESCC and deserves further investigation.Fig. 2Silencing ODC expression by shRNA suppresses anchorage-dependent and anchorage-independent ESCC cell growth. a ODC expression was analyzed by Western blot in KYSE450 and KYSE510 cells expressing shMock or shODC. b ODC activity was assessed as the release of L-[1-C14] ornithine and results are shown as percentage of the control group (set at 100%). c Anchorage-dependent cell growth was measured by MTS assay. d For anchorage-independent growth, cells were cultured in soft agar for 3 weeks and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. All data are shown as means ± S.D. of triplicate values from 3 independent experiments