Establishment of Stable MELK and FOXM1 Cell Lines

Plasmids including pCMV-Flag-His-puro-MELK (MELK), pCMV-Flag-His-puro-FOXM1 (FOXM1) and the empty vector pCMV-Flag-His-puro (Vector) were purchased from Transheep (Shanghai, China). For MELK overexpression, cells were transfected with 2 μg plasmids (MELK or Vector) for 48 h by using the Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Inc.) following the manufacturer's manual (23 (link)). Cells were then selected in the presence of 1.5 μg/mL puromycin (Sigma-Aldrich). After selection for 3 weeks, the stable colonies were picked up and expanded. For ectopic expression of FOXM1, the MELK-silenced KYSE30 and EC9706 cells were transfected with 2 μg plasmids (FOXM1 or Vector) for 48 h by using the Lipofectamine 2000 reagent. The cells were then exposed to puromycin (1.5 μg/mL) for 3 weeks.
Specific shRNAs targeting MELK (shMELK#1 and shMELK#2) or FOXM1 (shFOXM1#1 and shFOXM1#2) and a scramble shRNA (shNC) were obtained from Sigma-Aldrich. The indicated sequences were described in Table S1. Lentiviruses production and subsequently tranfection were performed as our previously described (23 (link)). The cells were then cultured with puromycin for 3 weeks to establish stable cells.