Plenti cmv mcs gfp sv puro

The GFP control plasmid was obtained from Addgene, pLenti-CMV-MCS-GFP-SV-puro (Addgene plasmid 73582).
The H2BE and H2B constructs were generated as previously described¹ and were provided by the Dulac lab. The H2B and H2BE coding sequences were then moved to the pLenti backbone through Gibson assembly. Primers were designed through NEB Builder to separately amplify H2B or H2BE and the pLenti backbone. Each primer contained a non-complementary region on its 5’-end that corresponded to approximately 10 bases on the other template. PCR amplification was performed with Q5 High-Fidelity DNA Polymerase (NEB M0491S). Following DPN1 digestion, fragments were ligated together using NEBuilder HiFi DNA Assembly Master Mix (NEB E2621S) and transformed into NEB 5-alpha Competent E. coli cells (E2621S). Plasmid sequence was verified through Plasmidsaurus long read sequencing.
The H2BE-I39V construct was generated by site-directed mutagenesis of the H2BE backbone using Pfu Turbo HotStart DNA polymerase (Agilent, 600322–51), and primers were created using the DNA-based primer design feature of the online PrimerX tool. Constructs were verified by Sanger sequencing.

All cell lines were maintained in minimum essential medium (MEM) (Gibco, 11095080) supplemented with 10% fetal bovine serum (FBS) (HyClone, SH30070.03) and 1% penicillin and streptomycin (Thermo Fischer, 15140122). MCF7, T47D, and BT474 parental cell lines were obtained from ATCC. All cell lines were authenticated and negative for mycoplasma contamination. When indicated, cells were cultivated in an ultralow adherence culture dish (Corning, 3262). MCF7 cell lines constitutively expressing SOD2 mutants were obtained by lentiviral transduction and selected using appropriate antibiotics. SOD2 mutants were cloned into lentiviral vector pLenti-CMV-MCS-GFP-SV-puro (29 (link)) (Addgene, 73582), by using an existing SOD2 sequence as previously described (13 (link)) with the addition of nuclear localization signal (NLS), nuclear exclusion signal (NES), or Flag-tag. HEK-293T cells were transfected using Lipofectamine 3000 (Invitrogen, L3000001) together with lentiviral packaging constructs 8.91 and VSVG. Stably transduced MCF7 cells were selected using 1 µM/mL puromycin (Gibco, A1113803).

Rat Drp1(−/17) and Drp1(16/17) cloned in pEGFP-C1 plasmids were kindly provided by Dr. Stefan Strack, University of Iowa ^{27 (link)}. Drp1 coding sequence were subcloned into pLenti-CMV-MCS-GFP-SV-puro (Addgene, 73582) with a N-terminal GFP tag and sequenced to confirm successful cloning of Drp1(−/17) and Drp1(16/17) plasmids. The plasmids were transfected in 293-FT cells for expression and lentiviral particle production. OVCA433 and SKOV3 cells were infected and transduced with the GFP vector control, GFP-Drp1(−/17) and GFP-Drp1(16/17) virus, selected for expression using 5ug/ml Puromycin for 1–2 weeks and sorted for GFP expression by flow cytometry to generate stable Drp1 overexpressing cells.