Human hepatocellular carcinoma cells HepG2, HLF and HLE, as well as HEK-293T (human embryonic kidney cell) were from Dr Shuguo Sun's lab. SK-Hep-1 cells (Procell # CL-0212) were purchased from Procell. HepG2 and SK-Hep-1 are P53-WT while HLF and HLE carry P53-G244A according to The Cancer Cell Line Encyclopedia. HepG2, HLF and HLE, as well as HEK-293T were cultured in Dulbecco’s Modified Eagle’s Medium (Gibco #12800-082) supplemented with 10% fetal bovine serum(PAN, Germany. #ST30-3302). SK-Hep-1 cells were cultured in MEM (Procell #PM150410) supplemented with 10% fetal bovine serum. All cell lines were maintained in 37 °C incubator with 5% CO2. Cycloheximide (Sigma #C7698), MG132 (MCE # HY-13259), or doxycycline hyclate (MCE #HY-N0565B) was added to cell culture at a final concentration of 50 μg/ml, 25 μM, and 1 μg/ml respectively where indicated. ML-323 was added at 8 μM, 16 μM, or 32 μM as specified in the text.
Sk hep 1 cells
Manufactured by Procell
SK-Hep-1 cells are a human liver adenocarcinoma cell line. They are adherent cells derived from the ascites of a patient with liver adenocarcinoma.
Automatically generated - may contain errors
3 protocols using sk hep 1 cells
1
Characterization of Hepatocellular Carcinoma Cell Lines
2
In Vitro Knockdown and Overexpression of FN-EDA and CD63 in Liver and Endothelial Cells
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LX-2 cells (HSC cell line) (Procell, Wuhan, CN) were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. Human umbilical vein endothelial cells (HUVECs) (ATCC, Rockville, MD, USA) were cultured in complete 1640 medium. SK-hep1 cells (Procell, Wuhan, CN) were cultured in complete MEM. Primary HSECs (ScienCell, Carlsbad, CA, USA) were cultured in complete ECM with 10% endothelial cell growth supplement (ScienCell, Carlsbad, CA, USA). For in vitro knockdown, FN-EDA-siRNAs, CD63-siRNA and control siRNA (Biosune, Jinan, CN) were transfected into HUVEC or LX-2 at 30 pmol/ml (12 well plate). For in vitro reoverexpression of CD63, the ORF of CD63 in pEnter and vector plasmid control (Biosune, Jinan, CN) were transfected at 1.5 μg/ml (12 well plate) one day after CD63-siRNA transfection each group were harvested 84 hours in total. Transient transfection was performed using Lipofectamine 3000 (Life Technologies; Gaithersburg, MD) according to the manufacturer’s protocol. The target interfering sequences are listed in Table 2 .
Target sequences for RNAi.
| Target sequence | |
|---|---|
| FN-EDA-siRNA1 | GGGACUCCUACCUUAGGUA |
| FN-EDA-siRNA2 | CCGUAAGUGACUACACCUA |
| CD63-siRNA | GCUGCCUCGUGAAGAGUAU |
3
Lentiviral Transduction of Endothelial Cells
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Human HSECs (ScienCell, Carlsbad, CA, USA) were cultured in ECM with 10% foetal bovine serum (FBS), 1% penicillin/streptomycin, and 10% endothelial cell growth supplement (ScienCell). Human umbilical vein endothelial cells (HUVECs) purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) were cultured in Hams F12k medium with 10% FBS and 1% penicillin/streptomycin. SK-HEP-1 cells (Procell, Wuhan, CN) were cultured in MEM with 10% FBS and 1% penicillin/streptomycin.
Lentiviral transduction tagged with GFP to downregulate or overexpress NRP-1 was performed in HSECs, HUVECs, and SK-HEP-1 cells according to the instructions from the manufacturer using 20 MOI of lentivirus-encoding target genes. All assays were performed 72 h after transfection. Transduction efficiency was confirmed via a confocal microscope. Protein/mRNA knockdown or overexpression was confirmed by western blot/qRT-PCR analysis.
Lentiviral transduction tagged with GFP to downregulate or overexpress NRP-1 was performed in HSECs, HUVECs, and SK-HEP-1 cells according to the instructions from the manufacturer using 20 MOI of lentivirus-encoding target genes. All assays were performed 72 h after transfection. Transduction efficiency was confirmed via a confocal microscope. Protein/mRNA knockdown or overexpression was confirmed by western blot/qRT-PCR analysis.
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