Plvx tight puro vector

The antibodies used in this study are listed in Additional file 1: Table S3. For generation of inducible GFP-STIL cell lines, the pLVX-Tight-Puro/STIL plasmid was generated by insertion of the full-length STIL cDNA into the pLVX-Tight-Puro vector (BD Bioscience). For the STIL promoter assay, the pGL3-STIL promoter-luciferase plasmid was constructed by insertion of PCR amplification of STIL promoter region into pGL3-basic vector (Promega). Four mutations of HIF1α DNA-binding sites in the STIL promoter (site1: nts − 195 to − 199; site2: − 894 to − 898; site3: − 1054 to − 1058; and site4: − 1235 to − 1239) were generated from pGL3-STIL wild-type promoter-luciferase construct using the QuikChange Lightning Site-Directed Mutagenesis Kit (Agilent; Cat# 210519). The cDNA constructs for various Flag-tagged STIL truncated mutants were described previously [43 (link)]. The pHA-HIF1α (ΔODD) construct was a gift from Dr. Muh-Hwa Yang [5 (link)]. The pGL3-SLUG promoter-luciferase construct was kindly provided by Dr. Cheng-Wen Wu [85 (link)]. The promoter-luciferase constructs of NANOG, SOX2, and POU5F1 were previously described [86 (link)]. For in vivo metastasis assay, the pEIF4G-As-Luc2 plasmid was a gift from Dr. Ruey-Hwa Chen [87 (link)].

The U2OS-based doxycycline inducible PLK4-myc cell line used in this study was as previously described^{40 (link)}. To obtain CEP120-null RPE1 cell lines inducibly expressing CEP120-GFP (WT or mutants), lentiviruses containing CEP120-GFP (WT or mutants) in the pLVX-tight-puro vector (BD Biosciences Clontech) were used to infect CEP120-null RPE1 Tet-On cells that stably express rtTA. The infected cells were selected with 10 μg/ml puromycin or were sterile-sorted by cell sorter (FACSAria, BD Biosciences) for GFP signal. The positive cells were selected and expanded as inducible lines. The expression of CEP120-GFP (WT or mutants) was induced by adding 1 μg/ml doxycycline to the culture medium.