Lipofectatmine2000

Manufactured by Thermo Fisher Scientific

Lipofectamine 2000 is a cationic lipid-based transfection reagent used to efficiently deliver nucleic acids, such as DNA and RNA, into mammalian cells. It forms lipid-nucleic acid complexes that facilitate the uptake and expression of the delivered genetic material within the target cells.

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Lab products found in correlation

Dulbecco s modified eagle s medium, by Corning (3 mentions) Rpmi 1640 medium, by Corning (3 mentions) Lapc4, by American Type Culture Collection (2 mentions) Lapc 4, by Corning (2 mentions) Iscove s modified dulbecco s medium, by Corning (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Mapk pathway luciferase reporter construct, by Qiagen (1 mentions) Dual luciferase reporter assay, by Promega (1 mentions) Lncap, by Corning (1 mentions) Lncap, by American Type Culture Collection (1 mentions) Plasmocin, by InvivoGen (1 mentions) Lookout mycoplasma pcr detection kit, by Merck Group (1 mentions) Dulbecco modified eagle medium (dmem), by Corning (1 mentions) Hek293t, by Corning (1 mentions) Hek293t, by American Type Culture Collection (1 mentions)

5 protocols using lipofectatmine2000

MAPK Pathway Activation by miR-4728

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MAPK signaling pathway activity was detected by luciferase reporter assay as previously described (Zhang et al., 2015 (link)). Ramos cells were seeded in a 96-well plate (2 × 104 cells per well). MAPK pathway-luciferase-reporter construct (SABiosciences, Frederick, MD) was co-transfected with mimic-miR-4728 or mimic-Ctrl with lipofectatmine 2000 (life technologies). The control vector of luciferase-reporter construct was used as a negative control (VC). The cells were collected for the dual luciferase reporter assay after 48 h culture (Promega). Results are calculated by dividing firefly luciferase activity by Renilla luciferase activity. Data were presented as mean ± SE (n = 3).

Wang W., Lin P., Yao H., Jia X., Sun J, & Feng L. (2018). MicroRNA-4728 serves as a suppressor and antagonist of oncogenic MAPK in Burkitt lymphoma. Saudi Journal of Biological Sciences, 25(5), 982-985.

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Targeting USP13 and AMFR with siRNA

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The siRNAs targeting USP13 were synthesized and transfected into cells by lipofectatmine 2,000 (Life Technologies) followed by qPCR or Immunoblot analysis. The siRNA sequences used in this study are as following: siUSP13#1: 5′-GCUCUGUCCUGUGUGGAAATT-3′; siUSP13#2: 5′-GCACGAAACUGAAGCCAAUTT-3′; siAMFR#1: 5′-GCAUGCACACCUUGGCUUUTT-3′; siAMFR#2: 5′-GGUUAUCUAUGGCUAGCUUTT-3′; siAMFR#3: 5′-GCACACUACCAACAUUCUUTT-3′; N.C.: 5′-UUCUCCGAACGUGUCACGUTT-3′.

Sun H., Zhang Q., Jing Y.Y., Zhang M., Wang H.Y., Cai Z., Liuyu T., Zhang Z.D., Xiong T.C., Wu Y., Zhu Q.Y., Yao J., Shu H.B., Lin D, & Zhong B. (2017). USP13 negatively regulates antiviral responses by deubiquitinating STING. Nature Communications, 8, 15534.

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Cell Culture and Transfection Protocols

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LNCaP, PC-3, LAPC-4 and 293T cells were purchased from ATCC (Manassas, VA). LNCaP, PC-3 cells were cultured in RPMI 1640 medium (Corning cellgro) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). LAPC-4 cells were cultured in Iscove's modified Dulbecco's medium (Corning cellgro) supplemented with 15% FBS (Thermo Fisher Scientific). 293T cells for lentiviral packaging were cultured in Dulbecco's modified Eagle's medium (Corning cellgro) supplemented with 10% FBS (Thermo Fisher Scientific). Transfections were performed using Lipofectatmine2000 (Thermo Fisher Scientific), following manufacturer's instructions. 75-90% transfection efficiencies were achieved.

Bai Y., Yang Y., Yan Y., Zhong J., Blee A.M., Pan Y., Ma T., Karnes R.J., Jimenez R., Xu W, & Huang H. (2019). RUNX2 overexpression and PTEN haploinsufficiency cooperate to promote CXCR7 expression and cellular trafficking, AKT hyperactivation and prostate tumorigenesis. Theranostics, 9(12), 3459-3475.

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Cell Line Characterization and Transfection

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The cell lines VCaP, PC-3, and LAPC-4 were purchased from ATCC (Manassas, VA) and authenticated via STR profiling. VCaP cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Corning cellgro) supplemented with 13% fetal bovine serum (FBS) (Thermo Fisher Scientific 10437028). PC-3 cells were cultured in RPMI 1640 medium (Corning cellgro) supplemented with 10% FBS (Thermo Fisher Scientific 10437028). LAPC-4 cells were cultured in Iscove’s modified Dulbecco’s medium (Corning cellgro) supplemented with 15% FBS (Thermo Fisher Scientific 10437028). Potential contamination mycoplasma was often checked using the Lookout Mycoplasma PCR Detection Kit purchased from Sigma-Aldrich. Cell culture medium was routinely supplied with Plasmocin (InvivoGen) to prevent mycoplasma contamination. Transfections were performed following manufacturer’s instructions with Lipofectatmine2000 (Thermo Fisher Scientific) or by electroporation using Electro Square Porator ECM 830 (BTX) with Mirus Ingenio solution. Approximately 75–90% transfection efficiencies were achieved.

Yang Y., Blee A.M., Wang D., An J., Pan Y., Yan Y., Ma T., He Y., Dugdale J., Hou X., Zhang J., Weroha S.J., Zhu W.G., Wang Y.A., DePinho R.A., Xu W, & Huang H. (2017). Loss of FOXO1 cooperates with TMPRSS2-ERG overexpression to promote prostate tumorigenesis and cell invasion. Cancer research, 77(23), 6524-6537.

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Transfection of Cell Lines for Molecular Studies

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The cell lines HEK293T and VCaP were purchased from ATCC (Manassas, VA). BPH-1 cells were kindly provided by Dr. Donald Tindall (Mayo Clinic). HEK293T cells were cultured in Dulbecco's modified Eagle's medium (Corning cellgro) supplemented with 10% FBS (Thermo Fisher Scientific, Cat# 10437028). VCaP cells were cultured in Dulbecco's modified Eagle's medium (Corning cellgro) supplemented with 13% FBS (Thermo Fisher Scientific, Cat# 10437028). BPH-1 cells were cultured in RPMI 1640 medium (Corning cellgro) supplemented with 10% FBS (Thermo Fisher Scientific, Cat# 10437028). Transfections were performed using Lipofectatmine2000 (Thermo Fisher Scientific) or by electroporation using an Electro Square Porator ECM 830 (BTX) with Mirus Ingenio solution, following manufacturer's instructions. Approximately 75–90% transfection efficiencies were routinely achieved, verified by expression of GFP co-transfected with the plasmid(s) of interest.

Blee A.M., Liu S., Wang L, & Huang H. (2016). BET bromodomain-mediated interaction between ERG and BRD4 promotes prostate cancer cell invasion. Oncotarget, 7(25), 38319-38332.

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