Pbabe hygro

For stable expression of wild-type BAP1 or inactive mutant p.C91S BAP1 in the BAP1-deficient M3 cell line UPMM2, retroviruses were generated in HEK-293GP cells cultured in DMEM medium (Thermo Fisher Scientific, 41965062) supplemented with 10% FBS and 1% Penicillin-Streptomycin. HEK-293GP cells were transfected with pVSV-G envelope plasmid [50 (link)] (Addgene, Watertown, MA, USA, 8454) and the constructs pBABE-hygro [51 (link)] (Addgene, 1765), pBABE-hygro-BAP1-HA [52 (link)] (Addgene, 154020) or pBABE-hygro-BAP1-C91S-HA [52 (link)] (Addgene, 154021), respectively, using TransIT-LT1 Transfection Reagent (Mirus, Madison, WI, USA, 731-0029) according to the manufacturer’s protocol. Virus-containing medium was collected and filtered through a 0.45 µm syringe filter (Corning, Wiesbaden, Germany, 431220). Culture medium of UPMM2 cells was replaced with filtered virus-containing medium for transduction and 48 h after transduction, cells were selected with 100 µg/mL hygromycin B (Santa Cruz, Dallas, TX, USA, sc-29067) for 1 week.

A truncated OVA (tOVA) encoding residues 212–386 was fused to GFP with a nuclear localization signal and cloned into pBABE-Hygro (Addgene, 1765) (88 (link)). Retrovirus generated using this plasmid was transduced into control and Sharpin-KO B16F1 prior to hygromycin selection. T cells from transgenic OT-1 mice were purified using the EasySep Mouse T cell isolation kit and cultured for 24 hours in wells coated with anti-CD3 and -CD28 to activate T cells. On the day of the experiment, target B16F1 cells were cocultured with activated OT-1 T cells with anti-TNF (50 μg/mL) mAb or IgG control. YOYO3 positivity was quantified as outlined above.

pBABE-Neo-hTERT (Counter et al., 1998 (link); Hahn et al., 1999 (link)) were obtained from Addgene. Full length lamin A, progerin and LAP2β were amplified from a cDNA library (human embryonic stem cell line H9). LAP2α was amplified from pTD15 (gift from Dr Roland Foisner, Vienna). cDNAs were cloned into retroviral vector pBABE-hygro (Addgene, Cambridge, MA) or doxycyclin-inducible lentiviral vector pTRIPZ (Open Biosystem, Singapore). Restriction sites and v5-tag were introduced during PCR amplification step. Retroviruses were generated and fibroblast cultures infected using standard procedures. Lentiviruses were generated according to manufacturer's protocol (OpenBiosystem). Doxycline-dependent expression was verified by western blotting, immunofluorescence and FACS analysis.

The vector pBABEhygro (Addgene) was introduced into MethA tumor cells by calcium phosphate precipitation to obtain hygromycin B resistant clones. D2SC/1 cells were similarly transfected with pBABEpuro. Transfected cells were cultured in growth medium containing 5 μg/ml puromycin or 100 μg/ml hygromycin (Life Technologies). To obtain fusion hybrid cells, 10⁷ hygromycin resistant MethA tumor cells were mixed with 5 × 10⁷ puromycin resistant D2SC/1 cells and briefly centrifuged. Cellular pellets were gently resuspended in 1 ml PEG 4000 (Merck, Darmstadt, Germany) containing 0.5 ml RPMI 1640 medium and incubated at 37°C for 90 s. Subsequently, 15 ml RPMI 1640 medium was added drop wise to the cells, then 20 ml RPMI 1640 medium with 10% fetal calf serum. After 5 min the cell suspension was centrifuged and cells were plated in RPMI 1640 medium and 10% fetal calf serum. After 24 h the fused cell hybrids were selected in the presence of 100 μg/ml hygromycin and 5 μg/ml puromycin. When colonies were developed, individual hybrid cell clones were isolated using cloning cylinders (Sigma-Aldrich) and 50 μl of trypsin-EDTA.

The following plasmids were purchased from Addgene: pBABE-hygro (#1765), pBABE-puro (#1764), mito-PAGFP (#23348), pclbw-mito TagRFP (#58425), pclbw-mitoCFP (#58426). The following primers were used to for site-directed mutagenesis by Gibson Assembly:

Mfn2^S378P F: (5′-CCGTTCGTCTCATCATGGATCCCCTGCACATCGCAGC-3′)

Mfn2^S378P R: (5′-GCTGCGATGTGCAGGGGATCCATGATGAGACGAACGG-3′)

Mfn2^A383V F: (5′-ATTCCCTGCACATCGCAGTTCAGGAGCAGCGGG-3′)

Mfn2^A383V R: (5′-CCCGCTGCTCCTGAACTGCGATGTGCAGGGAAT-3′)

Mfn2^Q386P F: (5′-GCACATCGCAGCTCAGGAGCCGCGGGTTTATTGCCTAGAAATGCGG-3′)

Mfn2^Q386P R: (5′-CCGCATTTCTAGGCAATAAACCCGCGGCTCCTGAGCTGCGATGTGC-3′)

Mfn2^C390F F: (5′-GGGTTTATTTCCTAGAAATGCGG-3′)

Mfn2^C390F R: (5′-CCGCATTTCTAGGAAATAAACCC-3′)

Mfn2^L710P F: (5′-GACATCACCCGAGATAATCCGGAGCAGGAAATTGCTGC-3′)

Mfn2^L710P R: (5′-GCAGCAATTTCCTGCTCCGGATTATCTCGGGTGATGTC-3′).