pCMV-TFEB-3X Flag plasmid was provided by Dr A Ballabio [28 (link)]. HEK293T cells were transfected with pCMV-TFEB-3x Flag plasmid using Lipofectmine™ 3000 (Invitrogen, L3000015) for 48 hours according to the manufacturer's protocol and then followed by the designated treatments.
Lipofectmine 3000
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lipofectamine 3000 is a transfection reagent designed to efficiently deliver nucleic acids, such as plasmid DNA, siRNA, or mRNA, into eukaryotic cells. It facilitates the uptake of these molecules into the cells, enabling gene expression or gene silencing studies.
Automatically generated - may contain errors
8 protocols using lipofectmine 3000
1
Overexpression of TFEB in HEK293T
2
Isolation of Mouse Primary Cardiomyocytes
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Neonatal hearts were isolated from 1–3 days old mice. After removing atria and large arteries, ventricles were minced and treated with collagenase buffer for 1 h at room temperature with gently shaking. The enzyme digestion was stopped by the addition of 10% FBS containing DMEM. Fibroblasts were removed twice by 1 h pre-plating. The cardiomyocytes were counted and plated on collagen-coated culture dishes and were maintained in 10% FBS in DMEM without antibiotics. Isolated mouse primary cardiomyocytes were cultured for 3 days and then transfected with Flag-tagged DPF3a WT and S348A mutant constructs using Lipofectmine 3000 (Invitrogen).
3
Transfection and EBSS treatment of HeLa cells
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According to the manufacturer's protocol, the shRNA (short hairpin RNA) targeting ANXA6 (Santa Cruz Biotechnology, sc‐29688‐V), the shRNA targeting Atg7 (Santa Cruz Biotechnology, sc‐41447‐V), GFP‐ANXA6 (GeneBio, EX‐A0185‐M98‐B), or GFP‐WIPI2 (WD repeat domain, phosphoinositide interacting 2; GeneBio, EX‐V0950‐M98‐B) were transfected into HeLa cells using Lipofectmine™ 3000 (Invitrogen, L3000015) and then followed by EBSS treatment.
4
CRISPR-Mediated Gene Deletion in KGN Cells
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The KGN cells were cultured in Dulbecco's modified Eagle Medium/F12 (DMEM/F12) medium containing 10% fetal bovine serum (FBS) in a six-well plate. For transient transfection experiments, cells were transfected with 2 μg Cas9 expression plasmid and 1 μg two sgRNA expression plasmid (0.5 μg each) for DNA fragment deletion or double digestion by lipofectmine 3000 (Invitrogen). After 36–48 h, the selection antibiotics blasticidin (5 μg/ml) and puromycin (1 μg/ml) were added to cells to select cells expressing Cas9 and sgRNA, and maintained in cells for 48 h. Five days later, the antibiotics were added to cells again and maintained for 48 h. After cell clones formed, the cells were taken for detecting deletion efficiency and functional assay. Medium was changed every 2–3 days. To detect deletion deficiency, primers spanning the two sgRNAs region, including the sgRNA locus were selected. The criterion of primer design was exhibited in Supplementary Figure S1a .
5
Transfection and Visualization of SARS-CoV-2 Replicon
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SARS-CoV-2 replicon/pYES1L plasmids were purified using the HiSpeed Plasmid Maxi Kit (QIAGEN, Hilden, Germany). 293 T cells were transfected with SARS-CoV-2 replicon/pYES1L plasmid using Lipofectmine 3000 (Invitrogen) according to the manufacturer’s protocol. Briefly, 293 T cells grown to 80% confluence were transfected with 2 μg of SARS-CoV-2 replicon/pYES1L plasmid with 4 μL Lipofectamine 3000 per well in 12-well plate or transfected with 200 ng of SARS-CoV-2 replicon/pYES1L plasmid with 0.4 μL Lipofectamine 3000 per well in 96-well plate. Thirty-six hours after transfection, the nuclei were stained with Hoechst (Invitrogen) and then fixed with 4% paraformaldehyde, fluorescence were observed and imaged by fluorescence microscopy, and cells were then harvested for Western blot analysis and luciferase activity assay.
6
Construct HIF-1α Overexpressing Glioma Model
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Construct the expression plasmid of HIF-1α sequence, and use the Opti-MEM (Invitrogen) and Lipofectmine 3000 (Invitrogen) to insert the plasmid containing HIF-1α cDNA into the primary glioma cells by liposome transfection according to the instructions to construct HIF -1α Overexpressing Primary Glioma Cell Model.
7
TUSC3 Expression in Glioma Stem Cells
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Human TUSC3 expression and empty vector constructs (bacterial stock) were obtained from Genscript (Piscataway, NJ, USA). Plasmid was purified using QuickLyse Miniprep Kit (Qiagen, Germantown, MD, USA). GSCs (2 × 105) were seeded in 6-well plate 24 h before transfection. Lipofectmine 3000 (ThermoFisher Scientific, Waltham, MA, USA) was used for transfection following manufacture’s protocol. Puromycin (2 μg/mL, InvivoGen, San Diego, CA, USA) was used for selection after transfection and the Puromycin concentration was then reduced to 1 μg/mL 5 days later for maintenance.
8
TUSC3 Rescue in Glioma Stem Cells
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Rescue experiments were performed in wild-type and TUSC3 knockout GSCs. In rescue groups, these GSCs were transiently transfected with either empty vector or full-length TUSC3 cDNA. Transfection was followed manufacturer’s instructions (Lipofectmine 3000, ThermoFisher Scientific).
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