Plasmid vector encoding miR-200c and empty pCMV vector were obtained from OriGene Company. Both vectors had Geneticin (G418) resistance as a marker for screening aims. SKOV3 cells were seeded in a 12 well-plate at a density of 0.5 × 106 cells/well and transfected with 1 μg of pCMV-miR-200c plasmid (miR-200c) or the corresponding empty vector (CTRL) using Lipofectamine 3000 (ThermoFisher Scientific), following the manufacturer’s instructions. 48 h post-transfection, cells were resuspended in fresh culture medium supplemented with 0.5 mg/ml G418 and distributed in 96 well-plate. The cells were kept under G418 selection for a couple of weeks in order to obtain G418 resistant clones. One clone from each transfection with pCMV empty vector and pCMV-miR-200c was obtained and used in our studies.
Pcmv vector
Manufactured by OriGene
Sourced in United States
The PCMV vector is a plasmid designed for gene expression in mammalian cell lines. It contains a cytomegalovirus (CMV) promoter, which drives high-level expression of the inserted gene of interest. The vector also includes a multiple cloning site for easy insertion of the target gene.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Merck Group (1 mentions)
Neomycin, by GE Healthcare (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Jetprime transfection reagent, by Polyplus Transfection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Plko 1 vector, by Addgene (1 mentions)
6 protocols using pcmv vector
1
Stable transfection of miR-200c in SKOV3 cells
2
Cloning and Mutagenesis of CIP2A Promoter
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pCGN‐vector and pCGN‐ATF6 (1–373) were purchased from Addgene (Cambridge MA, USA). pCMV‐vector and pCMV‐KIAA1524‐DDK were purchased from Origene (Rockville, MD, USA). The upstream region of the CIP2A promoter from the transcription start site (TSS) was cloned by PCR amplification from 293T cell genomic DNA and then inserted into the pGL3‐basic with BglII and HindIII digestions (forward primer: 5ʹ‐ATC GGC TAG CTG CTG ACA CAT CAT CAC AGA TG‐3ʹ; reverse primer: 5ʹ‐ATC GAG ATC TTC TCA GAA GCT TCC GGA TCC‐3ʹ). The mutagenesis for CIP2A promoter was performed using QuickChange site directed mutagenesis kit (Stratagene, La Jolla, CA, USA) with according to the manufacturer's instructions.
3
Silencing Cav1.3 and TRPV6 in Caco-2 Cells
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ADH1B (SKU: SC335414) and ALDH2 (SKU: MC217268) cDNA in pCMV vector were purchased from Origene (Rockville, MD). A vector-based short hairpin RNA (shRNA) method was used to silence CaV1.3 and TRPV6 gene expression in Caco-2 cells. Two targeting sequences each were chosen against the nucleotide sequence of human CaV1.3 (Gene ID: 776 CACNAID; NM_000720.2) and human TRPV6 (Gene ID: ECAC2; NM_018646) using Dharmacon siDesigner software: Target 1, CCGAATAGCTCCAAGCAAA (sequence position 290–308) and Target 2, GGAAGACCCAGAGATACA (sequence position 5697–5715) for CaV1.3, and Target 1, GACAAAGACTCAGTGGAA (sequence position 2207–2224) and Target 2, GAAACAGCGCTACACATA (sequence position 467–484) for TRPV6. To construct shRNA vectors, two pairs of oligonucleotides containing the antisense sequence, hairpin loop region (TTGATATCCG), and sense sequence with cohesive BamHI and HindIII sites were synthesized (Integrated DNA Technologies Inc., Coralville, IA).
4
Nucleolin Knockdown in DLBCL Cells
5
miR-330 Overexpression in CRC Cells
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Human CRC cells (HCT116 and SW480) were cultured in RPMI 1640, containing 10% FBS (GIBCO, Carlsbad, CA, USA) at 37°C with 5% CO2 condition. The cells were obtained from Pasture institute of Iran (Tehran, Iran) and all experiments were done in cell logarithmic phase. 2×105 HT116 and SW480 cells were transfected by PCMV-miR-330 and empty PCMV construct (as a negative control) at 50%-60% confluence. In order to transfect the constructs into the cells, jetPRIME transfection reagent (Polyplus, France) was used to transfer of PCMV vector (Origene, USA) to the cells according to the company’s instructions. The PCMV construct contains Geneticin resistant sequences for selecting the plasmid recipient cells. The selection of vector positive cells was done in two weeks by using geneticin antibiotic (50 mg/mL).
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Human CRC cells (HCT116 and SW480) were cultured in RPMI 1640, containing 10% FBS (GIBCO, Carlsbad, CA, USA) at 37°C with 5% CO2 condition. The cells were obtained from Pasture institute of Iran (Tehran, Iran) and all experiments were done in cell logarithmic phase. 2×105 HT116 and SW480 cells were transfected by PCMV-miR-330 and empty PCMV construct (as a negative control) at 50%-60% confluence. In order to transfect the constructs into the cells, jetPRIME transfection reagent (Polyplus, France) was used to transfer of PCMV vector (Origene, USA) to the cells according to the company’s instructions. The PCMV construct contains Geneticin resistant sequences for selecting the plasmid recipient cells. The selection of vector positive cells was done in two weeks by using geneticin antibiotic (50 mg/mL).
6
Functional Validation of SF3B4 and SPAG5
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We purchased SF3B4- and SPAG5-targeted small interfering RNAs (siRNA#1 and 2) from GenePharma (Shanghai, China). The SF3B4 and SPAG5 siRNAs were transfected into CC cells using Lipofectamine 2000 (Invitrogen, 11668–019). RNA and protein samples were extracted 48 h after transfection. The siRNAs used in this study are shown in Table S1 .
SF3B4 open reading frames (ORFs) were purchased from Vigenebio (Jinan, China, CH804949) and cloned into pCMV vector (OriGene, PS100069). SF3B4 short hairpin RNA (shRNA) designed based on siRNA#1 was synthesized by Sangon Biotech (Shanghai, China) and cloned into pLKO.1 vector (Addgene, 10879). The cell lines were generated by infecting with lentiviral particles for 24 h and selecting them with puromycin (2 µg/ml) for 7 days to obtain the stable transfection cell lines. The shRNA sequences are shown in TableS1 .
SF3B4 open reading frames (ORFs) were purchased from Vigenebio (Jinan, China, CH804949) and cloned into pCMV vector (OriGene, PS100069). SF3B4 short hairpin RNA (shRNA) designed based on siRNA#1 was synthesized by Sangon Biotech (Shanghai, China) and cloned into pLKO.1 vector (Addgene, 10879). The cell lines were generated by infecting with lentiviral particles for 24 h and selecting them with puromycin (2 µg/ml) for 7 days to obtain the stable transfection cell lines. The shRNA sequences are shown in Table
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