Plvx puro lentiviral vector

To knock down USP5 expression, short hairpin RNAs (shRNAs) targeting human USP5 mRNA (1#, GCTCTC AAGGGAATCTTTA; 2#, GGATATATGTACTGCCC AA; 3#, CCTACTATACTCCCAACTT; 4#, CCACTT GCTCAGTCGTCAA) and a specific scramble shRNA (NC) were cloned into pLKO.1 lentiviral vector (Addgene, Cambridge, MA, USA).
To ectopic express USP5, human USP5 cDNA was amplified with the following primers: forward, 5′ -CGGAATTC ATGGCGGAGCTGAGTGAGGAG-3′ and reverse, 5′-CGGGATCCTAGCTGGCCACTCTCT GG-3′, and cloned into pLVX-puro lentiviral vector (Clontech, Palo Alto, CA, USA).
For lentivirus production, 293T cells were transfected with lentiviral vector and packaging vectors using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). And 48–72 h later, viruses were collected from culture media, filtered and infected indicated ovarian cancer cell lines.

DNMT1 isoforms 1 and 3 were amplified using pcDNA3/myc-DNMT1 vector (obtained from addgene). DNMT1 isoforms were cloned in pcDNA3.1myc/his A (-) vector (Invitrogen #V855-20) at EcoR1 and Kpn1 restriction sites. DNMT1-isoforms in frame with uORF (upstream open reading frame) were generated by using a three-step cloning strategy: (i) where the sequence encoding uORF was amplified using H1299 genomic DNA and was cloned at Nhe1 and EcoR1 sites to generate pcDNA3.1-uORF construct, (ii) DNMT1 isoforms were cloned in the recombinant construct (pcDNA3.1-uORF) at EcoR1 and Kpn1 restriction sites, (iii) followed by deletion of EcoR1 restriction site located in-between uORF and DNMT1 isoforms, by site directed mutagenesis [using Q5® site-directed mutagenesis kit (NEB #E0554S)] to obtain the resultant constructs of pcDNA3.1 with uORF in frame with DNMT1 isoforms. For mitochondrial localization, DNMT1 isoforms were cloned into Sal1 and Not1 restriction sites of pCMV-myc-mito (Invitrogen #V822-20) vector with N-terminal mitochondrial targeting sequence (MTS) and C-terminal myc epitope. For stable cell line generation, DNMT1-isoform3 was cloned at EcoR1 and Xba1 restriction sites in plvx-puro lentiviral vector (Clontech #632164). The generated constructs were confirmed by sequencing, using ABI 3130xl genetic analyzer.

WIF1 overexpression was assessed using two different methods. (1) The WIF1 expression vector pcDNA3.1 vector was a generous gift from Professor Qian Tao, Cancer Center, Chinese University of Hong Kong. Transient transfections of MCL cells (10 × 10⁶ cells) were performed using the Electro square electroporator (BTX ECM 800, Holliston, MA, USA) (225 V, 8.5 msec, 03 pulse) using 1 μg of WIF1 or empty vector per million MCL cells. Cells were harvested at 48 h after transfection. The efficiency of target gene overexpression was assessed using Western blot. (2) Lentiviral particles were generated by transfecting the 293T packaging cell line (Clontech Laboratories, Inc., Fremont, CA, USA) with the WIF1 lentiviral vector (pLenti-GIII-CMV-Puro) (Applied Biological Materials Inc., Richmond, BC, Canada) or its control (pLVX-Puro lentiviral vector) (Clontech Laboratories, Inc., Fremont, CA, USA), according to the manufacturer’s suggestion. CMV sequencing primer 5′-CGCAAATGGGCGGTAGGCGTG-3′ in which WIF1 sequenced and verified. JeKo-1 and Mino cells were infected with lentiviral particles at 24 and 48 h. Forty-eight hours after the second infection, cells were washed with phosphate-buffered saline (PBS) and lysed using the previous Western blot protocol.

Preparations of B6Tert-1 cells with the human prostasin expression silenced using short interfering RNAs (siRNA) were described previously [30 (link)]. Construction of B6Tert-1 cell lines with a stable over-expression of prostasin (B6/Pro) or the vector alone (B6/Vec) was described previously, as well [30 (link)]. The expression of prostasin in the B6/Pro cells is under the control of the cytomegalovirus (CMV) promoter and is also regulated by the tet repressor, allowing the induction of prostasin expression upon the addition of tetracycline (1 μg/ml) in the culture medium. JIMT-1 cells stably over-expressing prostasin without the tet repressor (JIMT-1/Pro) or harboring the vector alone (JIMT-1/Vec) were constructed using the method described previously [30 (link)]. Transient expression of prostasin in the UROtsa cells was accomplished by using a lentivirus harboring the human prostasin cDNA (pLVX-Pro) in the pLVX-Puro lentiviral vector (Clontech Laboratories, Inc.). A lentivirus with the pLVX-Puro vector alone was used as a control.

For the stable overexpression of GDF15, a plasmid-encoding full-length GDF15 was cloned from pLenti-C-mGFP-P2A-Puro-GDF15 (Origene, Rockville, MD, USA) into the pLVX-puro lentiviral vector (Clontech). For stable transfection in HEK-293T (Clontech) cells, lentiviral vectors were co-transfected with the virus mix (Sigma) using Lipofectamine 3000 (Invitrogen). The supernatant medium containing the virus was harvested and concentrated using a Lenti-X-Concentrator (Clontech) and the virus was added to AGS and SNU216 cells with 5 µg/mL polybrene (Santa Cruz Biotechnology). Thereafter, the medium was replaced with fresh medium containing puromycin (Sigma), and cultured in the presence of puromycin for 2 weeks to identify puromycin-resistant clones.