Non-targeting siRNA (siNT) and a pool of 4 USF3 siRNA (siUSF3) were commercially obtained (GE Dharmacon, Lafayette, CO), optimized and transfected into HEK293T, 8505C, and Nthy-ori 3-1 cells respectively with Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). USF3 mRNA expression was evaluated by qPCR to confirm knockdown efficiency at different time points. Human USF3 cDNA was cloned into pCDNATM3.1/Hygro(+) vector (Thermo Fisher Scientific) with EGFP tag at c-terminal end of open reading frame (ORF). MISSIONTM USF3 shRNA (shUSF3) and non-targeting shRNA control (shNT) was purchased from Sigma-Aldrich. USF3-del1Q-GFP, USF3-del3Q-GFP, vector-GFP, shUSF3, and shNT plasmids were transfected into HEK293T cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Fresh medium was replaced 6 h post transfection. USF3-del1Q-GFP, USF3-del3Q-GFP, vector-GFP transfected cells were observed under a fluorescent microscope to confirm the transfection efficiency, and sorted by flow cytometry for the qRT-PCR experiment. While shUSF3 and shNT transfected cells were confirmed by qPCR on USF3 mRNA expression and selected by puromycin to generate stable cell lines.
Lipofectamine 3000 transfection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, China, Germany, United Kingdom, Australia, France, Italy, Japan, Netherlands, Canada
Lipofectamine 3000 is a transfection reagent designed to facilitate the delivery of DNA, RNA, or other macromolecules into mammalian cells. It is a proprietary formulation that enhances nucleic acid uptake and expression in a wide range of cell types.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (160 mentions)
Opti mem, by Thermo Fisher Scientific (83 mentions)
Dual luciferase reporter assay system, by Promega (77 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (68 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (62 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (58 mentions)
Opti mem medium, by Thermo Fisher Scientific (41 mentions)
Polybrene, by Merck Group (32 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (28 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (27 mentions)
Puromycin, by Merck Group (26 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (25 mentions)
Pspax2, by Addgene (23 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (23 mentions)
Streptomycin, by Thermo Fisher Scientific (22 mentions)
Penicillin, by Thermo Fisher Scientific (22 mentions)
Pmd2 g, by Addgene (21 mentions)
Trizol reagent, by Thermo Fisher Scientific (20 mentions)
Puromycin, by Thermo Fisher Scientific (19 mentions)
Si nc, by GenePharma (19 mentions)
2 370 protocols using lipofectamine 3000 transfection reagent
1
Evaluation of USF3 knockdown and overexpression
2
miRNA Mimic and Inhibitor Transfection
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miR-1a-3p mimics, inhibitors, and negative controls (NCs) were purchased from GenePharma (China). Transfection of the miRNA mimics, inhibitors, and negative controls was performed with Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific, United States) following the manufacturer's instructions. Transfection of the sFRP1 plasmid was performed with Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific, United States) and P3000. After 6 hours of transfection, the medium was replaced.
3
NF-κB and RELA 3'-UTR Luciferase Assay
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For the luciferase assay with NF-κB, pGL4.32 (luc2P/NF-κB-RE/Hygro) vector containing five copies of NF-κB response element (Promega) was used. The hDPCs were transfected with the reporter vector and miR-146b mimic or miR-146b NC using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and then stimulated with LPS (100 ng/mL). For the luciferase assay with RELA 3′-UTR reporter vector, synthesized RELA 3′-UTR containing wild-type or mutated hsa-miR-146b-5p target sequences (400 bps each; Eurofins Genomics, Ebersberg, Germany) were inserted into XhoI and HindIII sites of a pMIR-REPORT vector (Thermo Fisher Scientific). The reporter vectors along with miR-146b mimic or miR-146b NC were transfected into hDPCs via Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Transfected cells were lysed with a luciferase cell culture lysis reagent and the luciferase activity was measured using luciferase assay system (Promega) and a luminometer (Luminescence PSN; ATTO).
4
MicroRNA-3613-3p Modulation in Breast Cells
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MiRNA mimics and miRNA inhibitors were designed and synthesized by Guangzhou RiboBio (RiboBio, China). MDA-MB-231 and MDA-MB-468 cells were seeded at 20 × 104 cells per well into 6-well plates and transfected with 10 nM miRNA-3613-3p mimics, or the appropriate miRNA mimic control using Lipofectamine3000 transfection reagent (Thermo Fisher). MCF10A cells were seeded at 10 × 104 cells per well into 6-well plates and transfected with microRNA-3613-3p inhibitors at a final concentration of 10 nM or the appropriate miRNA inhibitor control using Lipofectamine3000 transfection reagent (Thermo Fisher). Total RNA and protein were collected for assay 48–72 h after transfection.
5
Visualizing Active RhoA and α-Catulin in MDCK Cells
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To visualize active RhoA, MDCK cells were transfected with GFP-AHPH-DM (Addgene #71368) construct using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific #L3000001) according to manufacturer’s instructions. To visualize α-catulin protein, MDCK cells were transfected with construct of pEGFPC1 vector with cloned mouse α-catulin cDNA upstream of GFP using Lipofectamine 3000 Transfection Reagent (Thermo Fisher Scientific #L3000001) according to manufacturer’s instructions. Both constructs were verified through sequencing. Fluorescence was examined under confocal microscope LSM 700 (Zeiss) 24 h after transfection.
6
Investigating miR-103 Modulation in 293T Cells
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The sequence for miR-103 mimic was 5'-AGCAGCAUUGUACAGGGCUAUGA-3', and the nonsense sequence for miR-103 mimic negative control (NC-mimic) was 5'-UUCUCCGAACGUGUCACGUTT-3'. The sequence for miR-103 inhibitor was 5'-TCATAGCCCTGTACAATGCTGCT-3' and the nonsense sequence for miR-103 inhibitor negative control (NC-inhibitor) was 5'-CAGUACUUUUGUGUAGUACAA-3'. The sequences were all designed and synthesized by Guangzhou RiboBio Co., Ltd. The 293T cells (China Center for Type Culture Collection) were transfected with 100 nmol/l miR-103 mimic or NC-mimic using Lipofectamine® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The 293T cells were cultured in DMEM (HyClone; Cytiva) with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc.) at 37˚C and 5% CO2 for 24 h. For miR-103 inhibitor groups, the 293T cells were transfected with 100 nmol/l miR-103 inhibitor and NC-inhibitor using Lipofectamine® 3000 transfection reagent for 12 h at 37˚C. The medium was changed after 12 h and the cells were incubated for additional 2 days.
7
PINE-TREE and RoT Transfection in HEK293T
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For PINE-TREE-based transfection, HEK293T cells were transfected in 24-well tissue culture plates 24 h after seeding, when cells were around 40% confluent. 300 ng PE (pEF1α-PE2), 100 ng pegRNA expression plasmid (PE-pegRNA(DT)-nsgRNA(DT)), and 100 ng pEF1α-BFP were transfected per well using 1 μL Lipofectamine™ 3000 transfection reagent (Thermo Fisher Scientific) and 0.75 μL P3000 reagent (Thermo Fisher Scientific). For RoT transfection, 300 ng PE (pEF1α-PE2), 100 ng pegRNA expression plasmid (PE-pegRNA(DT)-nsgRNA(DT)), and 10 ng pEF1α-GFP were transfected per well using 1 μL Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) and 0.75 μL P3000 reagent (Thermo Fisher Scientific). The medium was changed 24 h post-transfection. Cells were dissociated using Accutase 72 h post transfection and passed through a 0.40-μm filter before sorting.
8
Overexpression and Knockdown of TTK in EC Cells
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2 μg of TTK overexpression vector and the corresponding control vector (Origene, Rockville, MD, USA) or 2 μg of TTK-targeting shRNAs (sc-36758-SH, Santa Cruz Biotechnology) and a control shRNA (sc-108060, Santa Cruz Biotechnology) were used per well for 12-well plates to transfect Ishikawa and HEC-1 cells using the Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). Thermo Fisher Scientific provided the miR-21 mimic/inhibitor (30 nM) and the control mimic/inhibitor (30 nM), which were transfected into EC cells using the Lipofectamine 3000 transfection reagent.
9
Transfection of HeLa and PC12 Cells with RCOR2
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HeLa cells were transfected using pcDNA3.1 or pcDNA3.1-HA-RCOR2 (insert: NM_054048.4) using a ratio of 500 ng Plasmid DNA every 1 uL Lipofectamine 3000 transfection reagent (Thermo Fisher, #L3000075) and Opti-MEM reduced serum medium supplemented with GlutaMAX (Thermo Fisher, #51985034) according to manufacturer instructions. Cells were harvested 24 h after transfection and used for RT-qPCR, Western blot and or immunostaining. PC12 cells were transfected using pcDNA3.1-Myc-RCOR2 (insert: NM_173587.4) using a ratio of 500 ng Plasmid DNA every 1 uL Lipofectamine 3000 transfection reagent (Thermo Fisher, #L3000075) and Opti-MEM reduced serum medium supplemented with GlutaMAX (Thermo Fisher, #51985034) according to manufacturer instructions.
10
Investigating TRIM25's Impact on HL-1 Cell Viability
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To study the effect of TRIM25 on the HL-1 cells’ viability, HL-1 cells were infected with shrna -TRIM25, for 24 h using Lipofectamine™ 3000 Transfection Reagent (#L3000015, ThermoFisher, Shanghai, China) according to the manufacturer’s Reagent protocol.
Expression plasmids encoding p85α (encoded by Pik3r1), ubiquitin (Ub), TRIM25-WT, TRIM25-2EA, p85α-K506R, and p85α-K511R (p85α mutants with lys506 and lys511 changed into Arg were termed as K506R and K511R) were purchased from Genechem (Genechem, Shanghai, China). All plasmids were transfected with Lipofectamine™ 3000 Transfection Reagent (#L3000015, ThermoFisher, Shanghai, China) according to the protocal.
Adenovirus-driven ectopic expressing TRIM25 was achieved by infecting cultured cells at 50 MOI.
The reagents used and their commercial sources were indicated as follows: Doxorubicin (DOX) (selleckchem), cycloheximide (CHX) (selleckchem), MG132 (selleckchem), Tauroursodeoxycholic Acid (TUDCA) (selleckchem), Tunicamycin (TM) (MedChemExpress, Shanghai, China) and protein G agarose beads (#16–266, Merck Millipore, USA).
Expression plasmids encoding p85α (encoded by Pik3r1), ubiquitin (Ub), TRIM25-WT, TRIM25-2EA, p85α-K506R, and p85α-K511R (p85α mutants with lys506 and lys511 changed into Arg were termed as K506R and K511R) were purchased from Genechem (Genechem, Shanghai, China). All plasmids were transfected with Lipofectamine™ 3000 Transfection Reagent (#L3000015, ThermoFisher, Shanghai, China) according to the protocal.
Adenovirus-driven ectopic expressing TRIM25 was achieved by infecting cultured cells at 50 MOI.
The reagents used and their commercial sources were indicated as follows: Doxorubicin (DOX) (selleckchem), cycloheximide (CHX) (selleckchem), MG132 (selleckchem), Tauroursodeoxycholic Acid (TUDCA) (selleckchem), Tunicamycin (TM) (MedChemExpress, Shanghai, China) and protein G agarose beads (#16–266, Merck Millipore, USA).
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