Lncap fgc

The androgen-dependent human prostate carcinoma cell line LNCaP-FGC (ATCC CRL-1740, Rockville, MD, USA) was cultured in RPMI-1640 (Thermo Scientific, Waltham, MA, USA) supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA, USA) and penicillin/streptomycin at 37° C in 5% CO₂. The γRV vector, CL-SGN, was previously described [9 (link)]. The LV vector, LV-SGN, has SIN LTRs, a strong internal SFFV promoter driving an EGFP-neomycin fusion protein. Concentrated γRV and LV vector stocks pseudotyped with a vesicular stomatitis virus-glycoprotein (VSV-G) envelope were produced by polyethylenimine transient transfection of human embryonic kidney 293 (HEK 293T) cells using helper plasmids pLGPS (γRV vector), psPAX2 (LV vector) and the VSV-G envelope helper plasmid pMD2.G. Viral supernatant was filtered using 0.45 µm (γRV vector) and 0.22 µm (LV vector) filters (Pall Life Sciences, Cornwall, UK) and centrifuged for 18 h at 12,100 g. The viral supernatant was concentrated 100 fold. Viral titers were determined by transduction of HT1080 fibrosarcoma cells and analyzed for EGFP expression by flow cytometry.

LNCaP-FGC and PC-3 PCa cell lines were purchased from ATCC (Rockville, MD) in 2010 and cultured as described previously [3 (link)]. Dihydrotestosterone (DHT) was obtained from Amersham (Braunschweig, Germany). Bortezomib and MG132 were obtained from LC Laboratories (Woburn, MA, USA). Sea plaque agarose for soft agar experiments was purchased from Cambrex (Rockland, ME, USA). The plasmids bearing the HA-tagged OTUB1 allele and the C91S mutant have been described previously [27 (link)]. The functional screening was performed using siRNA pools purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For RhoA Knockdown shRNA pools from Santa Cruz Biotechnology (Santa Cruz, CA, USA) were used. OTUB1 individual siRNAs were purchased from Qiagen (Hilden, Germany) Transfections were performed using the Neon transfection system (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Stable PC3 cell lines were generated by transfecting the SureSilencing shRNA plasmid for human OTUB1 (Qiagen, Hilden, Germany) encoding the Neomycin resistant gene and the pGL4.17-[luc2/puro] (Promega, Madison, WI, USA), PC3-shOTUB1, or the same plasmid containing a scramble sequence which does not match any human gene, PC3-shControl. Expression vectors for WT-RhoA, Q63L-RhoA and DN-RhoA were described previously [39 (link)].

The androgen-dependent human prostate carcinoma cell line LNCaP-FGC (ATCC CRL-1740) was cultured in RPMI-1640 supplemented with 10% FBS (Atlanta Biologicals, Lawrenceville, GA) at 37°C in 5% CO₂. The LV shuttle vector, LV-SFFVEGFP, has self-inactivating long terminal repeats (LTRs), an internal spleen focus-forming virus promoter driving EGFP expression, and R6Kγ origin of replication and a neomycin phosphotransferase gene. Vesicular stomatitis virus glycoprotein pseudotyped vector stocks were made by PEI-mediated transfection of HEK-293T cells as previously described
[64 (link)]. Functional titers were determined by transduction of HT-1080 fibrosarcoma cells. Cells were cultured for 14 days post vector exposure prior to use in experiments.

Human prostate cancer cell lines LNCaP‐FGC, PC‐3 and DU145 were purchased from ATCC (Rockville, MD, USA). LNCaP cells had been passaged directly from original low‐passage stocks (2009), and we confirmed PC‐3, LNCaP and DU145 cell identity by STR profiling in 2010 and 2014 (Cellbank, Sydney). Cells were cultured in RPMI 1640 medium containing 10% v/v fetal bovine serum (FBS), penicillin–streptomycin solution and 1 mm sodium pyruvate. Cells were maintained at 37 °C in an atmosphere containing 5% CO₂. Chemicals were diluted as follows, with control wells treated with the appropriate vehicle controls: H‐Ser(Bzl)‐OH (BenSer; Bachem; diluted in H₂O); l‐γ‐glutamyl‐p‐nitroanilide (GPNA; MP Biochemicals; diluted in H₂O); bicalutamide (Astra Zeneca; diluted in DMSO). The [³H]‐l‐leucine and [³H]‐l‐glutamine uptake assays were performed as detailed previously 18, 32.