Transient miR-181a knockdown or overexpression was achieved with syn-hsa-miR-181a miScript miRNA mimic, control miR, anti-hsa-miR-181a miScript miRNA inhibitor, and miScript inhibitor negative control (Applied Biosystems). Transfections were performed with GenMute transfection reagent (SignaGen Laboratories, Gaithersburg, MD). pmiRZIP lentivector expressing anti-miR-181a or control sequence (SBI, Mountain View, CA) was used for permanent miR-181a knockdown experiments. Transduction-ready FIV-based pseudoviral particles were generated using pPACK-H1 Lentivector Packaging System together with 293TN cell line (SBI), at a titer of 1.06 × 109 IFU/ml. Control was Lenti-scramble Hairpin control pseudoviral particles at a titer of 1 × 109 IFU/ml. Cells were cultured in 12-well plates at a density of 1× 105/well for 1 day, infected by pseudoviral particles (using a multiplicity of infection of 100 viruses per cell) and cultured for 72 hrs, then selected for puromycin (5µg/ml) resistance for stable cell lines. Stably transduced cells were used for vitro and in vivo experiments. Cells were transfected with human regulator of G-protein signaling 16 cDNA (RGS16, NM_002928) cloned into a pCMV vector (pReceiver-M02) and negative control Vector (EX-NEG-M02) (GeneCopoeia, Rockville, MD) and selected for neomycin resistance.
Ex neg m02
Manufactured by GeneCopoeia
Sourced in United States
The EX-NEG-M02 is a laboratory instrument designed for nucleic acid extraction. It utilizes a magnetic bead-based method to effectively isolate DNA, RNA, or other nucleic acids from various sample types. The core function of this product is to provide a reliable and efficient means of nucleic acid purification for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Ex z0396 m02, by GeneCopoeia (3 mentions)
Lentiviral shrna pgipz vectors ns rhs4348, by Thermo Fisher Scientific (3 mentions)
αcd133 v2lhs 71816, by Thermo Fisher Scientific (2 mentions)
Genmute transfection reagent, by SignaGen (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Ls174t cells, by American Type Culture Collection (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
H1975, by Thermo Fisher Scientific (1 mentions)
H1299, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
H1299, by Shanghai Cell Bank (1 mentions)
H1975, by Shanghai Cell Bank (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Puromycin, by Takara Bio (1 mentions)
Geneticin, by Thermo Fisher Scientific (1 mentions)
8 protocols using ex neg m02
1
Transient and Stable Modulation of miR-181a
2
CD133 Expression Plasmid and shRNA
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Human cDNA CD133 expression plasmid (EX-Z0396-M02) and empty vector plasmid (EX-NEG-M02) were obtained from GeneCopoeia. Lentiviral shRNA pGIPZ vectors; NS (RHS4348) and αCD133 (V2LHS_71816) were obtained from Thermo Scientific. IKK (IKK-2 S177E S191E) and IKBα (pBabe-Puro-IKBalpha-mut (superrepressor)) plasmids were obtained from Addgene.
3
Overexpression of Bcl-2 Variant α in DU145 Cells
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OmicsLink™ Expression Clones for transcript variant α of Bcl-2 (EX-H3307-M02) and control vector (EX-NEG-M02) were purchased from GeneCopoeia (Germantown, MD, USA). DU145 cells were transfected using a combination of Nupherin™ (Enzo Life Sciences, Farmingdale, NY, USA) and Lipofectamine® 2000 (Life Technologies) when the cells reached 70–80% confluence in 24-well plates. First, 1 μg of plasmid DNA was mixed with 10 μg of Nupherin in 150 μl of Opti-MEM® (Life Technologies) for 15 min and then combined with 150 μl of Opti-MEM containing 1–4 μl of Lipofectamine 2000 for another 40 min at room temperature. The culture media were replaced with the transfection media, and the cells were briefly centrifuged at 100 × g and incubated for 4 h. The transfection media were then replaced with 1 ml of culture media. After overnight culture, the cells were subcultured in F25 flasks with culture media containing 500 μg/ml of G418. The transfected cells were then cloned by limiting dilution under G418 selection and were passaged many times (>20) to develop stable cell lines.
4
Modulating NF-κB Activity in Cells
5
Authenticated Cell Lines for CD133 Study
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Authenticated human embryonic kidney (HEK) 293 cells and human colon cancer cell lines (LoVo and LS174T cells) were purchased from ATCC, immediately prepared frozen cell stocks and stored in liquid nitrogen freezer. Cells were passaged for fewer than 6 months after resuscitation. Cell line authentication was performed by ATCC using short tandem repeat DNA profiles. All cell lines were routinely maintained in ATCC-recommended conditions. 293-CD133 cells overexpressing CD133 were established by transfection with CD133-expressing plasmid, pcDNA3.1-CD133 (CD133 cDNA cloned into pcDNA3.1, Invitrogen), and were maintained with G418 (600 μg/ml, Invitrogen). Cells were incubated in a 37°C and 5% CO2 environment under humidified conditions. Human cDNA CD133 expression plasmid (EX-Z0396-M02) and empty vector plasmid (EX-NEG-M02) were obtained from GeneCopoeia.
6
Overexpressing FOXM1 in NSCLC cell lines
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Human NSCLC cell lines, A549, H1299, and H1975 cells were purchased from Shanghai Cell Bank of the Chinese Academy of Sciences. A549 cells were cultured with DMEM medium and H1299 and H1975 cells were grown in RPMI 1640 medium. Both DMEM medium and RPMI 1640 medium were supplemented with 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin sulfate (100 μg/ml). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere. The empty plasmid EX-NEG-M02 (Genecopoeia) and human FOXM1-overexpression plasmid (Genecopoeia) were transfected into A549, H1299, and H1975 cells using Lipofectamine 3,000 reagent (Invitrogen #2024201, MA, United States), according to the manufacturer’s instructions. After that, the A549, H1299, and H1975 cells transfected with the plasmid were used in subsequent experiments.
7
Plasmid Transfection for Gene Expression
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The BimEL cDNA expression vector (EX-O0071-M029) and control vector (EX-Neg-M02) were purchased from GeneCopoeia, Inc. The vectors/plasmids were isolated using a commercially available midi-prep kit (cat. no. 12143; Qiagen GmbH) following the manufacturer's protocol. For transfections, cells were seeded in DMEM-F12 medium with 5% DCC FBS to yield ~50% confluence. Adherent cells were transfected with plasmids (4.0 µg) by using either x-fect reagent (cat. no. 631317; Takara Bio USA, Inc.) or lipofectamine LTX (11668-019; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Following a 16-24 h transfection at 37°C, cells were treated for various times at 37°C and harvested for analysis as described in figure legends.
8
Establishing Stable Cell Lines for CD133 Expression
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Human cDNA CD133 expression plasmid (EX-Z0396-M02) and empty vector plasmid (EX-NEG-M02) were obtained from GeneCopoeia. Lentiviral shRNA pGIPZ vectors; NS (RHS4348) and aCD133 (V2LHS_71816) were obtained from Thermo Scientific.
MIA PaCa-2 (ATCC) and stable MIA-derivatives were maintained in DMEM (Hyclone) containing 10% fetal bovine serum. S2-VP10 cells were cultured in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum. Stable clones were selected and maintained in Geneticin (Invitrogen) and Puromycin (Clontech) for MIA PaCa-2 and S2-VP10 derivatives, respectively.
MIA PaCa-2 (ATCC) and stable MIA-derivatives were maintained in DMEM (Hyclone) containing 10% fetal bovine serum. S2-VP10 cells were cultured in RPMI 1640 (Hyclone) supplemented with 10% fetal bovine serum. Stable clones were selected and maintained in Geneticin (Invitrogen) and Puromycin (Clontech) for MIA PaCa-2 and S2-VP10 derivatives, respectively.
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