Plenti c myc ddk ires puro

Pancreatic cancer cell lines used in this study (BxPC-3 [#CRL-1687), SW1990 [#CRL-2172], SU.86.86 [#CRL-1837], PANC-1 [#CRL-1469], Hs 766T [#HTB-134], CFPAC-1 [#CRL-1918], MIA PaCa-2 [#CRM-CRL-1420], AsPC-1 [#CRL-1682], Panc 03.27 [#CRL-2549], HPAF-II [#CRL-1997], and Capan-2 [#HTB-80]) were purchased from ATCC and maintained in RPMI-1640 with 10% fetal bovine serum (FBS). The identity of each cell line was confirmed by DNA fingerprinting via short tandem repeats at the time of mRNA and total protein lysate preparation using the PowerPlex 1.2 kit (Promega). Fingerprinting results were compared with reference fingerprints maintained by the primary source of the cell line.
Transient knockdown of CES2 was performed by transfecting the cells using the following siRNAs: siControl (Silencer Select Negative Control No. 1, Thermo Fisher Scientific) and siCES2 (s225041, s528; Thermo Fisher Scientific). Short-hairpin RNAs targeting human CES2 mRNA and cloned into the pLKO.1-puro vector were obtained from the human library MISSION® TRC-Hs 1.0 (Sigma–Aldrich, TRCN0000046965). For overexpression, CES2 was cloned into the pLenti-C-Myc-DDK-IRESPuro (OriGene) vector, and an empty vector was used as a control. Lentiviral infections were conducted using 293LTV cells (Cell Biolabs, Inc.).

Full-length Pdpn (NM_006474.4) was subcloned into the AsiSI and XhoI restriction sites of the lentiviral vector pLenti-C-Myc-DDK-IRES-Puro (Origene), and the sequence was confirmed by Sanger sequencing. The constructed product was co-transfected into HEK-293T cells with lentiviral packaging plasmids (pAX2 and pVSVG), and the lentiviruses were harvested at 72 hours after transfection. After filtering through 0.45-μm filters, the reconstructed lentiviruses were concentrated through ultracentrifugation (2 hours at 50,000×g) and purified on a gradient (20% sucrose, 2 hours at 46,000×g) as previously described [28] (link). Then the reconstructed lentiviruses were used to infect U87 and U251 cells for 72 hours, and 3-8 μg/ml puromycin was chosen to select the infected cells. Pdpn protein levels in these cells were determined by immunoblotting.
To express the wild-type and mutant IDH1 in U87 and U251 cells, the wild-type IDH1 and mutant IDH1 (R132H) were subcloned into the pLenti-C-Myc-DDK-IRES-Puro vector (Origene) and pRRLSIN-cPPT-CMV-IRES-Hygro vector (Eureka Biotech, China), respectively, and the lentiviruses were generated and purified as previously described [29] (link). After being infected, the U87 and U251 cells were selected with puromycin and/or hygromycin.

For cloning of lentiviral overexpression constructs, first, the wild-type cDNA of REN (renin) was cloned into pLenti-C-Myc-DDK-IRES-Puro (OriGene) plasmid by digestion with Asc I and Mlu I. For the lentivirus production, 5 × 10⁵ human embryonic kidney (HEK) 293T/17 cells were cultured in one well of a six-well plate in high-glucose Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS. Twenty-four hours after seeding, the HEK293T/17 cells were transfected using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) and 1.2 mg of pLenti-C-Myc-DDK-IRES-Puro-REN plasmid, 1.2 mg of psPAX2 (#12260, Addgene), and 0.8 mg of pMD2.G (#12259, Addgene) packaging plasmids. Forty-eight hours after transfection, the viral particle containing supernatant was collected, cleared by centrifugation at 500g for 10 min, and filtrated (45 mm; 09-720-005, Thermo Fisher Scientific). The viral titer was estimated using the Lenti-X GoStix Kit (#631244, Clontech). For viral transduction, Flp-In 293 T-Rex cells were infected with the virus at multiplicity of infection ~0.4 and ~1. Lentivirus-transduced cells were selected with puromycin (2 mg/ml) starting 2 days after viral infection. Expression of the C-terminal Myc-DDK–tagged REN protein was verified by Western blot using anti-MYC antibody.