Pgipz control

Melanoma, pancreatic and glioblastoma cells were infected with the lentiviral expression system for short hairpin RNA (shRNA) for USP5 silencing, pGIPZ Control and pGIPZ-USP5 [19 (link)] were obtained from Open Biosystems. HEK293T cells were transfected with the lentiviral packaging vectors pMD2. G and psPax2 (Addgene) together with the shRNA vectors to produce virus using Poly Fect as described by the manufacturer (QIAGEN). The medium was changed to DMEM with 10% fetal bovine serum and after 48 hours, and the viral supernatant was collected with 2 mL of viral supernatant containing 4 μg/mL of Polybrene (Sigma-Aldrich). Two days after infection, the medium was changed and 1 μg/mL of puromycin was added. After puromycin selection (5 days), viable cells were recovered; USP5 levels were examined by immunoblotting. Those with stable reduction of USP5 were used to assess to analyze apoptotic sensitivity to TRAIL and other agents.

Melanoma cells were infected with the lentiviral expression system for short hairpin RNA (shRNA) against human pLVX-Usp9x, kindly provided by Dr Dzwokai Ma (University of California, Santa Barbara)40 (link). For NRAS and control KD: pGIPZ Control, pGIPZ-NRAS-1, and pGIPZ-NRAS-2 were obtained from Open Biosystems. Open Biosystems TRIPZ control (clone ID: RHS4743) and TRPIZ human Usp9x (clone ID: V3THS320834) doxycycline-inducible shRNA vectors were also used in melanoma cells. Doxycycline at 1 μg ml⁻¹ was used to induce shRNA expression.
Ets-1 shRNA was kindly provided by coauthor, Dr Peter C. Hollenhorst (Indiana University, Bloomington, Indiana). HEK293T cells were transfected with the lentiviral packaging vectors pMD2.G and psPax2 (Addgene) together with the shRNA vectors to produce virus using PolyFect as described by the manufacturer (QIAGEN). The medium was changed to DMEM with 10% fetal bovine serum, and after 48 h, viral supernatant was collected. Viral supernatant containing 4 μg ml⁻¹ of Polybrene (Sigma-Aldrich) was added to each melanoma cell line. Cells with stable KD were selected with puromycin.

Melanoma cells were infected with the lentiviral expression system for short hairpin RNA (shRNA)-mediated BRAF and Usp5 knockdown and their control; pGIPZ Control, pGIPZ-USP5, and pGIPZ-BRAF were obtained from Open Biosystems. Knockdown of p53 was achieved with the following sense targeting sequence: p53; 5′-GACTCCAGTGGTAATCTAC-3′ cloned into pRetrosuperpuro (Oligoengine, Seattle, WA, USA). pBabe-puro-BRAF-V600E and pDEST-LTR-N-FLAG-HA-IRES-USP5 expression vectors was obtained from Addgene. HEK293T cells were transfected with the lentiviral packaging vectors pMD2.G and psPax2 (Addgene) together with the shRNA vectors to produce virus using PolyFect as described by the manufacturer (QIAGEN). The medium was changed to DMEM with 10% fetal bovine serum and after 48 hours, and the viral supernatant was collected. Viral supernatant containing 4 μg/mL of Polybrene (Sigma-Aldrich) was added to each melanoma cell line. After puromycin selection, Usp5 stable knockdown, overexpressing or control cells were used for analysis.