HepG2 cells were seeded for 24 h, and transfected with Lipofecatmine 2000 and pcDNA3.1-HBx (2 µg) or pcDNA3.1-HBV (2 µg) or empty vector, pcDNA3.1 (2 µg) (Invitrogen, CA, USA) according to the manufacturer’s instruction. In the HBx and HBV plasmid transfection experiments, cells were used for RNA extraction 48 h post-transfection. In the HBx-siRNA experiment, HBx-siRNA or its negative control was co-transfected with pcDNA3.1-HBx or pcDNA3.1-HBV plasmid in HepG2 cells, and HBx-siRNA or its negative control was transfected in HepG2.2.15 cells; RNA were extracted 24, 48, and 72 h post-transfection. For the miR-19a inhibitor, miR-122 and miR-223 mimics experiments, miR-19a inhibitor, miR-122 and miR-223 mimics or their negative controls were co-transfected with pcDNA3.1-HBx in HepG2 cells. For the PTEN overexpression, cyclin G1 and—cymc silence experiment, pcDNA3.1-PTEN, c-myc-siRNA or cyclin G1-siRNA, or their negative controls were co-transfected with pcDNA3.1-HBx in HepG2 cells; for the rescue experiment, miR-19a inhibitor + PTEN-siRNA, miR-223 mimics + pcDNA3.1-cyclin G1, or miR-122 mimics + pcDNA3.1-c-ymc were co-transfected with pcDNA3.1-HBx in HepG2 cells.
Lipofecatmine 2000
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 2000 is a lipid-based transfection reagent used for the delivery of nucleic acids, such as DNA and RNA, into a variety of cell types. It facilitates the uptake of genetic material into the target cells, enabling gene expression studies and related research applications.
Automatically generated - may contain errors
Lab products found in correlation
Sirnas, by Thermo Fisher Scientific (2 mentions)
Hbx sirna, by Thermo Fisher Scientific (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Inhibitor nc, by RiboBio (1 mentions)
Si nc, by Thermo Fisher Scientific (1 mentions)
Mir 520b, by RiboBio (1 mentions)
Si nc, by OriGene (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Emt antibody sampler kit, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Signalway Antibody (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Wizard pcr clean up kit, by Promega (1 mentions)
12 protocols using lipofecatmine 2000
1
HBx and HBV Transfection Experiments in HepG2 Cells
2
Regulation of MLK3 by miR-520b
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miR-520b mimics(miR-520b), inhibitor(inh), miR-NC, inhibitor-NC(inhNC) were synthesized and purified by RiboBio (Guangzhou, China). The concentration of miR-520b, inh or inhNC was 100nM. Small interfering RNA targeting MLK3 (Si-MLK3) and negative control (Si-NC) were obtained from OriGene Technologies (Rockville, MD, USA). The concentration of Si-NC and Si-MLK3 was 100nM. Lipofecatmine 2000 (Invitrogen, USA) was used as transfection reagent as manufacture suggested. The experiments were done 48h after transfection unless clarified.
3
Antisense Oligonucleotide Knockdown of TOB1-AS1
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Three Affinity Plus® ASOs were synthesized by Integrated DNA Technologies (IDT), with two targeting TOB1-AS1 and one non-targeting negative control. The ASO sequences were:
Non-targeting ASO (NC): 5’-GGCTACTACGCCGTCA-3’
TOB1-AS1 ASO1: 5’-GCCGATTTGGTAGCTA-3’
TOB1-AS1 ASO2: 5’-CTGCGGTTTAACTTCC-3’
The ASOs were transfected into PATU-8988S and Panc 10.05 cells with Lipofecatmine™ 2000 (Invitrogen, 11668019) using reverse-transfection method according to IDT protocol (https://www.idtdna.com/pages/products/functional-genomics/antisense-oligos ) with a final ASO concentration of 9 nM. Cells were transfected in 6-well plates and incubated for 48 hours to reach 60% confluence before RNA extraction or Transwell assay.
Non-targeting ASO (NC): 5’-GGCTACTACGCCGTCA-3’
TOB1-AS1 ASO1: 5’-GCCGATTTGGTAGCTA-3’
TOB1-AS1 ASO2: 5’-CTGCGGTTTAACTTCC-3’
The ASOs were transfected into PATU-8988S and Panc 10.05 cells with Lipofecatmine™ 2000 (Invitrogen, 11668019) using reverse-transfection method according to IDT protocol (
4
Knockdown of USP14 in Prostate Cancer
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This assay was performed as we previously described.39 (link) To knock down USP14 expression in prostate cancer cells, siRNA or shRNA targeting human USP14 were synthesized and purchased from Santa Cruz Biotechnology Inc.. siRNA or shRNA with non-specific sequences were used as control scrambled RNA. Different siRNAs and shRNAs were transfected separately into cells using Lipofecatmine 2000 (Invitrogen, Carlsbad, CA, USA) reagent and the medium was replaced 6 h after the transfection.
5
HBx siRNA Knockdown in HBV Genotype D
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HBx plasmid -pCXN2-HBx of genotype D was gifted by Dr. Tatsuo Kanda, Japan. 1.3 fold full length HBV genome (genotype D) cloned into pUC 19 vector was a generous gift of Dr. Mashashi Mizokami, Japan. RNA was extracted 48 hours post transfection. HBx-siRNA
[24 (link)] was used to produce small interfering RNAs (siRNAs) targeting HBx mRNA (genotype D) (Ambion, TX, USA). siRNA duplexes with non-specific sequences were taken as negative control (NC) for siRNA experiments. siRNA transfection were carried out using Lipofecatmine 2000 (Invitrogen, CA, USA) reagent and medium was replaced 6 hour after transfection. RNA was extracted at 24, 48 and 72 hours post siRNA treatment.
[24 (link)] was used to produce small interfering RNAs (siRNAs) targeting HBx mRNA (genotype D) (Ambion, TX, USA). siRNA duplexes with non-specific sequences were taken as negative control (NC) for siRNA experiments. siRNA transfection were carried out using Lipofecatmine 2000 (Invitrogen, CA, USA) reagent and medium was replaced 6 hour after transfection. RNA was extracted at 24, 48 and 72 hours post siRNA treatment.
6
HepG2 Lipofectamine Transfection Protocol
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Transfection was performed using Lipofecatmine 2000 (Invitrogen) following manufacturer’s instructions. Briefly, twenty four hours prior to transfection 5 × 105 HepG2 cells were seeded into a six well plate. Cells were transfected with two doses - 1 μg and 2 μg of pCXN2-HBx plasmid, 1.3 fold HBV plasmid (puC19-1.3 HBV) and empty vector. In case of HBx and HBV plasmid transfection, after 48 h, cells were used for RNA extraction. For siRNA experiments RNA were extracted 48 h post transfection.
7
HBx and HBV Plasmid Transfection
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Transfection was performed using Lipofecatmine 2000 (Invitrogen) following manufacturer’s instructions. Briefly, twenty four hours prior to transfection 5 × 105 HepG2 cells were seeded into a six well plate. Cells were transfected with two doses - 1 μg and 2 μg of pCXN2-HBx plasmid, 1.3 fold HBV plasmid (puC19-1.3 HBV) and empty vector. In case of HBx and HBV plasmid transfection, after 48 hours, cells were used for RNA extraction. For siRNA experiments RNA were extracted 24, 48 and 72 hours post transfection.
8
HBx gene silencing via siRNA
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HBx plasmid -pCXN2-HBx was gifted by Dr. Tatsuo Kanda, Japan. 1.3 fold full length HBV genome cloned into pUC 19 vector was a gift of Dr. Mashashi Mizokami, Japan. HBx-siRNA [27 (link)] was used to produce small interfering RNAs (siRNAs) targeting HBx mRNA (Ambion, Texas, USA). siRNA duplexes with non-specific sequences were taken as negative control (NC) for siRNA experiments. siRNA transfection was carried out using Lipofecatmine 2000 (Invitrogen, Carlsbad, CA, USA) reagent and medium was replaced 6 h after transfection. RNA were extracted following different time interval.
9
Antibody-Based EMT Pathway Analysis
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Antibodies against CBX8 (catalog No. ab182627), ZEB1 (catalog No. ab124512) and ZEB2 (catalog No. ab138222) were purchased from Abcam (Cambridge, MA, USA). Epithelial–mesenchymal transition (EMT) antibody sampler kit (catalog No. #9782) and phospho-β-catenin antibody (catalog No. #9561) were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-β-actin antibody (catalog No. 21800) was from Signalway Antibody LLC (College Park, Maryland, USA). Lipofecatmine 2000 (catalog No. 11668019) for cell transfection and Trizol (catalog No. 15596018) for RNA extraction were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All other reagents were from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise noted.
10
Cloning and Luciferase Assay for Murine Slc2a4 Enhancer
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The region of interest (ROI) chr11:69838231-69838257 (mm39) was amplified from genomic murine DNA using primers with restriction enzyme overhangs (sequence in Table S3 ). The insert was purified from an agarose gel using the Wizard PCR Clean-Up Kit (Promega, Madison, WI, USA) and cloned into the pCpGfree-mcs_v2 vector (InvivoGen, San Diego, CA, USA) at the HindIII (New England BioLabs, USA) and NcoI (New England BioLabs, USA) restriction sites using the QuickLigation™ Kit (New England BioLabs, Ipswich, MA, USA). Correct insertion was checked by sequencing using the vector_seq primer (Supplementary Table S2 ). A total of 50 ng of the pCpGfree-mcs_v2-Slc2a4 insert vector was co-transfected with 1 ng of reference Renilla vector (pRL; Promega, USA) per well of a 96-well plate into HEK293T cells using 0.5 µL of Lipofecatmine 2000 (ThermoFisherScientific, USA) and incubated for 24 h at 37 °C and 5% CO2. For measuring firefly luciferase and renilla signal, the Dual-Glo® Luciferase Assay System (Promega, USA) was used according to manufacturer’s instructions.
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