The LMNA plasmid was purchased from Sino Biological (Beijing, China). The LMNA gene was then subcloned and inserted into a PLVX-puro lentivirus vector (Clontech, Shiga, Japan). The PLVX-shRNA1 lentivirus plasmid for LMNA shRNA was constructed as previously described [35 (link)]. The LMNA shRNA sequence was as follows: 5′-CTGACTTCCAGAAGAACA-3′. Lentivirus lacking the shRNA insert was used as a control.
To establish LMNA knockdown or overexpression of LMNA cells, 293T cells were transfected with the target plasmid along with psPAX2 and PMD2G packing plasmids with Lipo8000 Transfection Reagent (Beyotime, Shanghai, China, C0533). Next, 293T cell supernatants were collected and mixed with fresh medium to infect HCC827 or HCC827/ER cells along with 2 μg/mL polybrene (Beyotime, C0351). After 24 h, the supernatants were replaced with fresh medium containing puromycin (Beyotime, ST551) at a concentration of 5 μg/mL. The puomycin-resistant cells were isolated and used for further experiments.
To establish LMNA knockdown or overexpression of LMNA cells, 293T cells were transfected with the target plasmid along with psPAX2 and PMD2G packing plasmids with Lipo8000 Transfection Reagent (Beyotime, Shanghai, China, C0533). Next, 293T cell supernatants were collected and mixed with fresh medium to infect HCC827 or HCC827/ER cells along with 2 μg/mL polybrene (Beyotime, C0351). After 24 h, the supernatants were replaced with fresh medium containing puromycin (Beyotime, ST551) at a concentration of 5 μg/mL. The puomycin-resistant cells were isolated and used for further experiments.
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