Plvx tight puro

The human RTTN cDNA (accession number, BC156291) was purchased from the IMAGE clone consortium (IMAGE clone No. 100061722) and subcloned into pEGFP-N2 (BD Biosciences Clontech) or pFlag-CMV2 (Sigma-Aldrich). To generate the GST-fusion constructs, the cDNAs encoding various potion of RTTN were fused in-frame to the GST-encoding sequence in the pGEX4T vector (GE Healthcare). The RTTN mutant constructs were generated by site-directed mutagenesis using a QuikChange kit (Stratagene). The expression constructs encoding various GFP-tagged RTTN mutant proteins were generated using pcDNA4/To/myc-His-A (Invitrogen) or pLVX-Tight-Puro (BD Biosciences Clontech) vectors. Sequencing was used to confirm all generated plasmids. The cDNAs encoding full-length CP110, H2B and STIL were obtained by RT-PCR using total RNAs from human HEK293T cells and subcloned in-flame into a pEGFP-C1 (BD Biosciences Clontech) or a pLVX-Tight-Puro (BD Biosciences Clontech) vector. The cDNA constructs for GFP-tagged full-length STIL, SAS-6, and CPAP and various Flag-tagged STIL truncated mutants were from ref. ^{8 (link)}. The cDNA constructs containing various CEP295 fragments were from ref. ^{19 (link)}.

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)].