The eukaryotic expression vectors encoding Flag- or Myc-tagged proteins or untagged proteins were generated by inserting polymerase chain reaction (PCR)–amplified fragments into pcDNA3 (Invitrogen). Prokaryotic plasmids encoding GST- or His-fusion proteins were constructed in pGEX-KG (Amersham Pharmacia Biotech) and pET32a (Novagen), respectively. The target sequences of siRNAs and/or shRNAs for PES1 and hTERT were listed in table S1. Because of the extremely low expression level of GST-hTERT protein in E. coli, we optimized the coding sequence of hTERT (table S2) and cloned the optimized sequence into the pGEX-KG vector. The Wnt signaling reporter plasmids TOPFlash and FOPFlash were gifts from X. Yan (Nanchang University). The NF-κB-Luc reporter was a gift from X. Yu (Beijing Institute of Basic Medicine). Lentiviral vectors for gene overexpression were obtained by inserting PCR-amplified gene fragments into pCDH-EF1-MCS-T2A-Puro (System Biosciences). Lentiviral shRNA vectors were constructed by cloning shRNA fragments into pSIH-H1-Puro (System Biosciences). Lentiviruses were produced by cotransfection of HEK293T cells with recombinant lentivirus vectors and pPACK Packaging Plasmid Mix (System Biosciences) using MegaTran reagent (Origene) and were used to infect target cells according to the manufacturers’ instructions.

Anti-Myc (sc-40HRP), β-actin (sc-47778HRP), and anti-PES1 for immunoblot (sc-166300) were purchased from Santa Cruz Biotechnology; anti-Flag (A8592) and anti-Flag M2 agarose (A2220) were obtained from Sigma-Aldrich; anti-GST (RPN1236) and anti-His (27471001) antibodies were purchased from GE Healthcare Life Sciences; anti-PES1 for IHC (A300-902A) was purchased from Bethyl Laboratories; anti-hTERT (abx120550) was purchased from Abbexa; anti-hTERT (ab32020), anti-DKC1, and anti-WDR12 (ab95070) were purchased from Abcam; anti-Hsp90 (60318-1-Ig) and anti-p23 (15216-1-AP) were purchased from Proteintech; anti–c-Myc (D84C12) was purchased from Cell Signaling Technology.

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