Pcmv6 ddk myc

AGS and MKN1 gastric cancer cells lines were cultured as described in Supplementary information. Complete NKX6.3, Skp2, c-Myc and mutant β-catenin^S37A-cDNA was cloned into the expression vector pCMV6-Myc-DDK (Origene), pcDNA3.1 (Invitrogen) and pQNCX2 (gift from Dr. Eek Hoon Jho, University of Seoul, Seoul, South Korea). We generated stable NKX6.3 transfectants of AGS and MKN1 cells, AGS^NKX6.3 and MKN1 ^NKX6.3, stably expressing human NKX6.3, as well as mock transfectants, AGS^Mock and MKN1^Mock cells, as described previously and in the Supplemental information (Yoon et al., 2015 ). Stable expression of NKX6.3 was confirmed in AGSN^KX6.3 and MKN1^NKX6.3 cells by western blot analysis.

Doxorubicin (Dox, Cat#44583), vinblastine (VIN, Cat#V1377), vincristine (VCR, Cat#V8388) and verapamil (Cat#V4629) were purchased from Sigma-Aldrich (St. Louis, MI, USA) and prepared following manufacturer’s instructions. CD133 expression plasmid pCMV6-CD133-Myc-DDK, its control vector pCMV6-Myc-DDK, and transfection reagent TurboFectin 8.0 were purchased from Origene (Rockville, MD, USA). Human specific CD133 short interfering (si) RNA (oligonucleotide ID# HSS113055), scrambled control siRNA oligonucleotide (12935-200), and siRNA transfection reagent Lipofectamine® RNAiMAX were purchased from Invitrogen (Life Technologies, Grand Island, NY, USA).

HEK-293 T cells were purchased from the Shanghai Cell Bank (GNHu17, Shanghai, China) and cultured in high-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, 11,960,044) with 10% fetal bovine serum (FBS; FB15015; Clark Biosciences, USA), 1% Glutamax (Invitrogen, 35,050,061), and 1% Sodium Pyruvate 100 mM Solution (Invitrogen, 11,360,070) at 37℃ and 5% CO₂.
The wild-type (WT) plasmid (pCMV6-SPOCK2-Myc-DDK) and control (empty) plasmid (pCMV6-Myc-DDK) were purchased from OriGene (Rockville, MD, USA). ΔKAZAL (mut1) plasmid (pCMV6-ΔKAZAL-myc-DDK), ΔSPARC_EC (mut2) plasmid (pCMV6-ΔSPARC_EC-myc-DDK), and ΔΔ (mut3) plasmid (pCMV6-ΔΔ-myc-DDK), containing both knockout parts of the ΔKAZAL and ΔSPARC_EC plasmids, were obtained from TSINGKE Biological Technology (Beijing, China). Lipofectamine 3000 (Invitrogen, Waltham, MA, USA) was used for plasmid transfection according to the manufacturer’s instructions.