A double-stranded oligonucleotide corresponding to the human MSK1 sequence (5′- ATTTCCAAACAAGTCATAGGT-3) was synthesized and cloned into the pBS/U6 vector (Addgene, Cambridge, MA, USA). The plasmid was then used to construct the short hairpin RNA (shRNA) for MSK1 downregulation. Meanwhile, pcDNA 3.1 vector (Addgene) was used for construction of the MSK1 expression plasmid. In addition, the human CREB sequence was cloned into the pcDNA 3.1 vector and then the construct CREBser133 by site-directed mutagenesis. Transient transfection was performed with Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) as protocols.
Pbs u6 vector
Manufactured by Addgene
Sourced in China
The PBS/U6 vector is a plasmid used for the expression of short hairpin RNA (shRNA) or other small RNA species in mammalian cells. It contains a human U6 promoter for driving the expression of the target RNA sequence.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3.1 vector, by Addgene (2 mentions)
Padtrack vector, by Addgene (2 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
Adeno x rapid titer kit, by Takara Bio (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Psilencer 2.1 u6 hygro, by Thermo Fisher Scientific (1 mentions)
Het 1a cell line, by American Type Culture Collection (1 mentions)
5 protocols using pbs u6 vector
1
Modulating MSK1 and CREB Expression
2
Overexpression and Knockdown of Asxl1 in Mammalian Cells
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For overexpression in mammalian cells, Asxl1 adenovirus was generated using the pAdEasy system45 (link). Flag-tagged mAsxl1 was subcloned into the pAdTrackCMV plasmid and recombined with the pAdEasy adenovirus backbone plasmid in E. coli BJ5183. QBI-293A cells were transfected with recombinant plasmid, and the virus was amplified. The virus titer was measured using an Adeno-X Rapid Titer Kit (Clontech). For KO in mammalian cells, recombinant adenovirus-expressing shRNA was prepared. The duplex DNA effective for both human and mouse ASXL1 (Supplementary Table S5 ) was digested with Notl and subcloned into the digested PBS/U6 vector (Addgene). The U6 promoter-driven shASXL1 was excised and ligated into the pAdtrack vector (Addgene). The pAdTrack-U6 shASXL1 obtained was recombined with pAdEasy-1 by transformation in E. coli BJ5183. Recombinant adenovirus was produced by transfecting the recombinant plasmid into QBI-293A cells. Infection and KO efficiency were monitored by GFP fluorescence and RT-qPCR, respectively. To evaluate the optimal multiplicity of infection (MOI) for maximal infection and transgene expression, 60-mm dishes containing 2×105 A549 cells were infected with adenovirus at MOIs of 50, 100, and 200 for 24 h. Adenoviral infection efficiency was assessed based on GFP expression. Asxl1 expression in A549 cells was evaluated by RT-PCR and WB.
3
Glioma Cell Silencing Targets
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U-138MG and U-251MG human glioma cell lines, and HEK-293 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Small interfering RNA Runx1 (siRunx1, 5′-ATCACTGGCGCTGCAACAAGAC-3′; 5′-GGATGTT-CCAGATGGCACTCT-3′) were designed and synthesised by Sangon Biotech Co., Ltd (Shanghai, China) and cloned into the pBS/U6 vector (Addgene, Cambridge, MA, USA). A siSTAT3 (5′-GGACAATATCATTGACCTT-3′; 5′-CCACTTTGGTGTTTCATAA-3′) was designed and synthesised by Sangon Biotech Co., Ltd, and cloned into the pBS/U6 vector. Additionally, pcDNA3.1 vector (Addgene) was used for construction of Runx1 expression plasmid. U-138MG and U-251MG cells were transfected with siRunx1, siSTAT3, and their negative control (siNC) via FuGENE Transfection Reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions.
4
Generation of ASXL1-targeting shRNA Adenovirus
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The synthetic oligonucleotides used for the depletion of ASXL1 using small hairpin RNA (sh RNA) are shown in Supplementary Table 3 . Each duplex was formed and digested with HindIII and BamHI and ligated with the digested pSilencer 2.1-U6 hygro (Ambion). pSilencer hygro luciferase was used as a control (shLuc). For the knockdown in WI-38 cells, recombinant adenovirus expressing shRNA was prepared. The duplex DNA effective for both human and mouse ASXL1 was digested with NotI, and subcloned into the digested PBS/U6 vector (Addgene). The U6 promoter-driven shASXL1 was cut out and ligated with pAdtrack vector (Addgene). The pAdTrack-U6 shASXL1 obtained was recombined with pAdEasy-1 by transformation in E. coli BJ5183. Recombinant adenovirus was produced by transfecting the recombinant plasmid into QBI-293A cells. Infection and knockdown efficiency was monitored by GFP fluorescence and RT-qPCR, respectively.
5
Regulation of ESCC Cell Lines
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Human esophageal epithelial cell line (Het-1A) and ESCC cell lines (TE-1, KYSE-30, EC109 and EC9706) were used in the present study. Het-1A cell line was obtained from the American Type Culture Collection (Manassas, VA, United States) and TE-1 cell line was purchased from Chinese Academy of Medical Sciences (Beijing, China). In addition, KYSE-30 and EC109 cell lines were derived from CoBioer (Nanjing, China) and EC9706 cell line was gotten from Shanghai Huiying (Shanghai, China). KYSE-30 or EC109 cells were transfected with the small interfering RNA (siRNA) targeting lnc-MCEI (si-MCEI), miR-6759-5p mimics, miR-6759-5p inhibitor at 50 % confluence. Lnc-MCEI and negative control (NC) hairpin RNA (shRNA) sequences were purchased from GenePharma (Shanghai, China) and cloned into the pBS/U6 vector (Addgene) for lnc-MCEI knockdown. Additionally, the IGF2 expression plasmids construction and transient transfection were performed as the protocol previously described 19 .
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