Construction of stable OLC1-overexpressing cell lines, and the effects of OLC1 overexpression on KYSE150 cells.
(A) To investigate OLC1 expressions among ESCC cell lines (EC9706, KYSE 510, KYSE 150, KYSE450 and KYSE180), the relative protein levels of OLC1 from these cell lines were examined by western blotting analysis. (B) KYSE150 cells were transfected with pEGFP-N1-OLC1 and pEGFP-N1, and stable, positive clones were selected using with G418 (400 µg/mL) from candidate clones. Two positive clones were designated as OLC-1 and OLC-2, the null control colony was designated KYSE150/GFP. Confirmed by western blotting, the expressed GFP-OLC1 fusion protein (68 kDa) was detected in OLC-1 and OLC-2, but not in KYSE150/GFP cells (C) mRNA levels of OLC1 in OLC-1, OLC-2 and KYSE150/GFP cells, assessed by RT-PCR. (D) Cell proliferation curves were calculated after counting the numbers of OLC-1, OLC-2 and KYSE150/GFP cells for 5 days. (E) The numbers of colonies of each cell line were counted, and then compared. All experiments were repeated three times. Significance was determined by the Student’s t test. * = P<0.05; ** = P<0.001.