Plvx ires neo lentivirus vector

The NCOA4 short hairpin RNA (shRNA) was subcloned into pLVX-shRNA lentivector (Takara, Dalian, China) for NCOA4 knockdown. The sequences for NCOA4 silencing were as follow: 5’-GATCCGTCAGCAGCTCTACTCGTTATTTTCAAGAGAAATAACGAGTAGAGCTGCTGATTTTTTG-3’, and 5’-AATTCAAAAAATCAGCAGCTCTACTCGTTATTTCTCTTGAAAATAACGAGTAGAGCTGCTGACG-3’. The exogenous expression of FTH protein was performed according to our previous publication [23 (link)]. Briefly, human full-length FTH cDNA was obtained from Sino Biological (Beijing, China), and subcloned into pLVX-IRES-Neo lentivirus vector (Takara, Dalian, China) by Seamless Cloning kit (TransGen Biotech, Beijing, China). The recombinant lentiviral plasmids were verified by sequencing and co-transfected with pMD2G, pSPAX2 into 293 T cells according to the instructions of Lipofectamine 3000 (L3000008, Invitrogen) to produce recombinant lentivirus. The stable cell lines with NCOA4 silencing or FTH overexpression were obtained via lentiviral transfection and antibiotics screening. The expression level was confirmed by Western blot.

LLC-MK2-GCaMP6s P2Y1 knock in lines were generated by lentiviral transduction of the human P2Y1 receptor. The P2Y1 cDNA clone was purchased from GenScript (Piscataway, NJ, USA) and subcloned into pLVX-IRES-Neo lentivirus vector (Takara Bio) and packaged by the BCM Vector Development Core. LLC-MK2-GCaMP6s cells were transduced with MOI 10 in 10 µg/mL polybrene and at 72 h post-transduction selected using 500 µg/mL G418 (22 (link)). The presence of functional P2Y1 was verified by Ca²⁺ imaging for a Ca²⁺ response to 25 nM ADP.

Full-length FTH was ordered from Sino Biological (Beijing, China) and amplified by PCR (AP131-11, TransGen Biotech), then subcloned into pLVX-IRES-Neo lentivirus vector (Takara, Dalian, China) by ClonFast Seamless Cloning kit (Obio, Nanjing, China). The recombinant lentiviral plasmid was verified by sequencing. The recombinant lentiviral plasmid was co-transfected with pMD2.G, pSPAX2 into 293T cells to produce recombinant lentiviral. After 72 h of transfection, the supernatants were collected, centrifuged, and filtered through a 0.4 μm filter. To generate stably transfected cell lines, PANC1 cells (8 × 10⁴) were seeded in 24-well plates. After 12 h, the culture medium was removed, and cells were transfected with the corresponding lentivirus. After 2 days of transfection, 1 mg/mL G418 was added for selection for 7 days. Then the stable cells were maintained in 0.2 mg/mL G418. The transduction efficiency was evaluated by western blot analysis.