The HCV core expression plasmid, pCMV-3 × HA1-core, encodes the full-length HCV core (genotype 1b) downstream of three copies of the influenza virus hemagglutinin (HA) epitope [19 (link)]. The pCMV-3 × HA1-p16, encoding full-length HA-tagged p16, was described previously [20 (link)]. Scrambled (SC) shRNA, p53 shRNA, and PA28γ shRNA plasmids were purchased from Santa Cruz Biotechnology. Plasmid pCH110, encoding the Escherichia coli β-galactosidase (β-Gal) gene under the control of the SV40 promoter, and pCMV6 PSME3, encoding full-length human Myc-DDK-tagged PA28γ, were purchased from Pharmacia (Cat. No. 27-4508-01) and OriGene (Cat. No. SC321554), respectively. The E2F1-luc and pCMV p53-WT were gifts from Dr. Chang-Woo Lee (Sungkyunkwan University, Suwon, Korea). The pHA-Ub, encoding HA-tagged ubiquitin, was kindly provided by Y. Xiong (University of North Carolina at Chapel Hill, USA).
P53 shrna
Manufactured by Santa Cruz Biotechnology
Sourced in United States
P53 shRNA is a laboratory reagent designed for the specific knockdown of the p53 gene expression. It functions as a short hairpin RNA (shRNA) that targets the p53 mRNA, leading to its degradation and reduced protein production.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3.1 p53 plasmid, by Hanbio Biotechnology (2 mentions)
Scrambled sc shrna, by Santa Cruz Biotechnology (1 mentions)
Cycloheximide (chx), by Santa Cruz Biotechnology (1 mentions)
Puromycin, by MP Biomedicals (1 mentions)
Anti ubiquitin, by Cell Signaling Technology (1 mentions)
Anti p mdm2, by Cell Signaling Technology (1 mentions)
Anti atf6, by Cell Signaling Technology (1 mentions)
Anti bip, by Cell Signaling Technology (1 mentions)
Anti xbp1, by Cell Signaling Technology (1 mentions)
Anti pdi, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Salubrinal, by Enzo Life Sciences (1 mentions)
Anti puromycin, by Cell Signaling Technology (1 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Anti eif2α, by Cell Signaling Technology (1 mentions)
Anti p eif2α, by Cell Signaling Technology (1 mentions)
10 protocols using p53 shrna
1
Recombinant Plasmid Expression of HCV Core and p16
2
Plasmids and Constructs for HBx Studies
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The plasmid pCMV-3 × HA1-HBx (HA-HBx) encodes full-length HBx (genotype D) downstream of the three copies of the influenza virus haemagglutinin (HA) [56 (link)]. The 1.2-mer WT HBV replicon containing 1.2 units of the HBV genome (genotype D) and its HBx-null counterpart [44 (link)] were kindly provided by W. S. Ryu (Yonsei University, Seoul, Republic of Korea). The HBV core promoter/enhancer reporter construct, pHBV-luc, was previously described [44 (link)]. The plasmid RC210241 (Cat No. 003049), encoding the human NTCP, was obtained from OriGene (Rockville, MD, USA). The plasmid pSiah-1-Myc WT, encoding Myc-tagged Siah-1, has been previously described [45 (link)]. Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat No. sc-29435) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Siah-1 shRNA (Cat No. SHCLND-NM003031) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Plasmid pCH110 (Cat No. 27-4508-01), encoding the Escherichia coli β-galactosidase (β-Gal) gene, was obtained from Addgene (Watertown, MA, USA). The pCMV p53 encoding WT p53 was a gift from Chang-Woo Lee (Sungkyunkwan University, Suwon, Republic of Korea). The pHA-Ub encoding HA-tagged Ub was kindly provided by Y. Xiong (University of North Carolina at Chapel Hill, Chapel Hill, NC, USA).
3
Establishing Stable Cell Lines with Targeted Knockdowns
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DACH1 shRNA, p53 shRNA, and SLC25A37 shRNA were obtained from Santa Cruz Biotechnology. The eukaryotic expression vector3 (pcDNA).1-DACH1 plasmid, pcDNA3.1-P53 plasmid, and pcDNA3.1-SLC25A37 plasmid were purchased from Hanbio (Shanghai, China). P53 plasmids with different domains were cloned and inserted into the pcDNA 3.1-myc vector. VA-Lip-Control-shRNA, VA-Lip-SLC25A37-shRNA, VA-Lip-P53-shRNA, and VA-Lip-DACH1-shRNA were prepared according to previous reports.8 (link)–10 (link) According to the manufacturer’s protocols, shRNA and plasmids were transfected by Lipofectamine 3000. Stable clones were selected in 2 µg/mL puromycin for 4 weeks, and a single clone was isolated using a limited dilution technique.
4
Reprogramming Werner Syndrome Fibroblasts to Induced Pluripotent Stem Cells
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We reprogrammed WS fibroblasts with high-titer polycistronic lentivirus-expressing human OCT4, SOX2, KLF4, and c-MYC (hSTEMCCA) (Somers et al., 2010 (link)). Four days after transduction, cells were transferred to MEF feeders in iPSC culture medium supplemented with HDAC and TGF-β RI kinase inhibitors (Millipore, cat. no. CS204423, CS204420) for 12 days. WS iPSC colonies started to appear 2 weeks after transduction. Colonies were picked from day 30 to 40 and expanded on MEF feeders in hESC medium containing 80% KO Dulbecco’s modified Eagle’s medium (DMEM), 20% KSR, 1% nonessential amino acids, 1% GlutaMAX, 0.1 mM β-mercaptoethanol, and 10 ng/ml bFGF. iPSCs were characterized by expression of pluripotency markers NANOG, SOX2, OCT4, SSEA3, SSEA4, TRA1-60, and TRA1-81 using immunofluorescence staining. iPSC pluripotency was tested by injecting cells into SCID mice at the kidney and testis capsules and harvested for histologic analysis (Applied Stem Cells). Karyotyping was performed by the WiCell Cytogenetics Lab. To generate telomerized and p53 knockdown cells, we transduced WS fibroblasts with retrovirus-expressing hTERT (Addgene plasmid 1773) or p53 shRNA (Santa Cruz) and selected by 100 μg/ml hygromycin B or 1 μg/ml puromycin. Stable cultures were reprogrammed by hSTEMCCA as described above.
5
Comprehensive Protein Regulation Analysis
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Cycloheximide was from Santa Cruz Biotechnology. Actinomycin-D was from Sigma. MG132 was from Selleck Chemical. Puromycin was from MP Biomedicals. Kainic acid and Thapsigargin were from Alomone Lab. Pifithrin-α was from Adipogen Corporation. Salubrinal was from Enzo Life Sciences. Dimethyl sulfoxide (DMSO) was from Fisher Scientific. DMSO was used as a vehicle in this study. The antibodies used in this study were purchased from Santa Cruz Biotechnology (anti-Mdm2, anti-Lamin A/C, anti-HSP-90), GenScript Corporation (anti-Gapdh), Millipore (anti-Puromycin), and Cell Signaling (anti-ubiquitin, anti-p-Mdm2, anti-p53, anti-BiP, anti-PDI, anti-PERK, anti-ATF6, anti-XBP1s, anti-eIF2α and anti-p-eIF2α). HRP-conjugated secondary antibodies were from Santa Cruz Biotechnology and Cell Signaling. The lentiviral control non-target shRNA and p53 shRNA were from Santa Cruz Biotechnology.
6
Targeted Silencing of BRD7, p53, and SLC25A28
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BRD7 shRNA (sc-92998, sc-141741), p53 shRNA (sc-44218, sc-45917), SLC25A28 shRNA (sc-90800, sc-149448), and control shRNA were obtained from Santa Cruz Biotechnology. The pcDNA3.1-BRD7 plasmid (NM_001108440.1, NM_ 013263.5), pcDNA3.1-P53 plasmid (NM_030989.3, NM_000546.5), pcDNA3.1-SLC25A28 plasmid (NM_00110 9515.1, NM_031212.4) and control pcDNA3.1 vector were purchased from Hanbio (Shanghai, China). The different p53 domains were constructed by PCR and cloned into pcDNA3.1-myc vector. The resulting plasmid was verified by sequencing. Transfections were performed with Lipofectamine™ 3000 (Invitrogen, L3000008) according to the manufacturer's instructions. VA-Lip-BRD7-shRNA, VA-Lip-P53-shRNA, VA-Lip-SLC25A28-shRNA and VA-Lip-Control-shRNA were prepared according to our previous reports [12 ,13 (link)]. In brief, 5 mg VA was added into 50 μl DMSO to form VA solution. 280 nmol VA solution and 0.14 μmol lipotrust solution (Hokkaido System Science, LEO-01) were mixed by vortexing in a 1.5-ml tube at 25 °C. 12.24 nmol BRD7 shRNA, p53 shRNA, SLC25A28 shRNA or control shRNA was added into VA-Lip solution with stirring at 25 °C. The VA-Lip solution was filtered. Fractions were collected and the material trapped in the filter was reconstituted with PBS to achieve the desired dose for in vivo use.
7
Plasmid Construction and Reporter Assays
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The plasmid pCMV-3 × HA1-HBx (HA-HBx), which encodes HBx (genotype D) downstream of influenza virus haemagglutinin (HA) tag (YPYDVPDYA), was previously described [17 (link)]. The 1.2-mer wild-type (WT) HBV replicon, which contains 1.2 units of the HBV genome (1.2-mer WT; genotype D), and its HBx-null counterpart (1.2-mer HBx-null) were previously described [28 (link)]. The HBV core promoter/enhancer reporter, pHBV-luc, was previously described [28 (link)]. The plasmid pSiah-1-Myc WT encoding Myc-tagged Siah-1 was previously described [29 (link)]. The plasmid RC210241 (Cat No. 003049) encoding human sodium taurocholate cotransporting polypeptide (NTCP) was purchased from OriGene (Rockville, MD, USA). Scrambled (SC) short hairpin RNA (shRNA) (Cat No. sc-37007) and p53 shRNA (Cat. sc-29435) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Siah-1 shRNA (Cat No. SHCLND-NM003031) was obtained from Sigma-Aldrich (St. Louis, MO, USA). The plasmid pCH110 (Cat No. 27-4508-01) encoding the Escherichia coli β-galactosidase (β-Gal) gene was purchased from Addgene (Watertown, MA, USA). pCMV p53-WT and pHA-Ub were kindly provided by Dr. C.-W. Lee (Sungkyunkwan University, Suwon, Republic of Korea) and Dr. Y. Xiong (UNC-Chapel Hill, NC, USA), respectively.
8
Evaluating siRNA and shRNA Effects on Cell Proliferation
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Cells were transfected according to manufacturer’s instructions using Lipofectamine RNAiMAX (Invitrogen) and 9 nM siRNA. To control for non-specific siRNA effects, a pool of non-targeting control siRNAs (ON-TARGET plus non-targeting, Thermo Scientific) was used, also at 9 nM. siRNAs for E7 (GGACAGAGCCCAUUACAAU), p53 and Myc were purchased from Dharmacon. Cell numbers were counted every 24 hours for up to 4 days. Lentiviruses with Myc shRNA, p53 shRNA, E6E7 shRNA or GFP shRNA were purchased from Santa Cruz, GUMC-395 cells were infected according to the manufacturer’s protocol. 5 × 104 infected cells were plated and numbers of the attached viable cells were counted every 4 days, the curve was plotted as cell numbers versus days after plating.
9
Transfection and Silencing of Tumor Genes
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pcDNA3.1 p53, pcDNA3.1 SMAR1 and pcDNA3.0 Cox-2 or SMAR1-shRNA (300 pmole/million cells),and control pcDNA3.0 vectors (2 µg/million cells) were introduced into exponentially growing cancer cells using lipofectamine-2000 (Invitrogen, CA) according to the protocol provided by the manufacturer. Stably expressing clones were isolated by limiting dilution by selection with G418 (400 µg/ml; Cellgro, USA) and puromycin (1 µg/ml; Cellgro, USA) for 14 days, and cells surviving this treatment were cloned and assessed for p53, SMAR1 and Cox-2 by immunoblotting. For endogenous silencing of specific genes, cells were transfected with 300 pmol of HIF-1α-/Cox-2 -siRNA (Santa Cruz, CA) and p53 shRNA (Santa Cruz, CA) using lipofectamine-2000 for 12 h. The mRNA and protein levels were determined by RT-PCR and western blotting.
10
Plasmid and siRNA Transfection Protocol
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The empty plasmid, CCDC106 plasmid, ATF4 siRNA (Origene, Rockville, MD, USA) and CCDC106 siRNA, Scramble siRNA, p53-shRNA, Scramble shRNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used in this study. Transfection using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was carried out according to the manufacturer’s protocol.
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