Pcmv neo bam p53 r175h

A fragment containing the Δ40p53 open reading frame (ORF) was amplified from HepG2 cDNA using KOD PlusNeo polymerase (TOYOBO; Tokyo, Japan) and the following primer set: forward, 5′-GGATCCCAAGCAATGGATGAT-3′ and reverse, 5′-TCAGTCTGAGTCAGGCCCTT-3′. A fragment of the Δ40p53 ORF carrying the R175H or R273H mutation was amplified from pCMV-Neo-Bam p53 R175H (plasmid 16436, obtained from Addgene) or pCMV-Neo-Bam p53 R273H (plasmid 16439, obtained from Addgene), respectively. Each fragment was inserted into a pBabe-puro vector or a pLXSN vector (Clontech Laboratories, Inc., Mountain View, CA). The retroviral plasmids were packaged in 293T cells using the pCL10A vector. Viral supernatants were harvested 96 h after transfection and filtered before infection. Experimental HCC cells were infected with retroviruses in the presence of 8 μg/ml Polybrene (Sigma-Aldrich). Antibiotic selection (puromycin; 2 μg/ml for pBabe-puro or neomycin; 800 μg/ml for pLXSN) was begun 48 h after infection and continued for at least 3 days.

RPMI1640 medium, Opti-modified Eagle’s medium (Opti-MEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), RNase A, leupeptin, aprotinin, phenylmethylsulfonylfluoride, and Triton X-100 were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). The pCMV-Neo-Bam p53-R175H, pCMV-Neo-Bam p53-R248W, pCMV-Neo-Bam p53-R273H, and pCMV-Neo-Bam p53 wild-type were obtained from Addgene (Cambridge, MA, USA). The antibodies against anti-p53, anti-Rad21, anti-S1PR1, anti-THBS1, and anti-β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The protein G PLUS-agarose was also obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The human CD133 promoter was cloned into the pG5 luciferase reporter vector, and the C/G to A/T mutation in the consensus p53-binding site was generated by site-direct mutagenesis. The human CD133 clone was obtained from the Korea Human Gene Bank (Medical Genomics Research Center, KRIBB, Daejeon, South Korea) and subcloned into the retroviral pBABE vector. All of the constructs were confirmed via sequencing. A set of lentiviral shRNAs against CD133 was purchased from Open Biosystems (shCD133-1(TRCN0000062143), shCD133-2(TRCN0000062144), and shCD133-3(TRCN0000062146)) (Open Biosystems, Huntsville, AL, USA). pCMV-Neo-Bam p53 R175H was a gift from Bert Vogelstein (Addgene plasmid # 16436, Cambridge, MA, USA).