Phoenix ampho cells

Retrovirus encoding the tet regulated NRAS or KRAS gene was packaged in Phoenix-AMPHO cells obtained from ATCC. The medium containing virus was filtered with 0.45 PVDF filters followed by incubation with the target cells for six hours. After this incubation, cells were cultured in virus free medium for two days. Then the cells were selected with Puromycin (2ug/mL) or Hygromycin (250ug/mL) for three days. The positive infected cells were further sorted with GFP marker after overnight exposure to 1ug/mL doxycycline (Sigma Aldrich). The NRAS gene construct included three consecutive FLAG tags in the N-terminus, and the KRAS gene construct included one FLAG tag in the N-terminus.

STHdhQ7 and STHdhQ111 cells were obtained from HD Community BioRepository Collection (New Jersey, USA) and cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Mediatech, Inc. VA, USA), 1% glutamine and 1% penicillin/streptomycin at 33°C. Early passages of STHdh (less than 20 passages) were used in the studies. Primary fibroblasts, GM04693 from a HD individual (onset at age 41 years) and AG02222 from a normal subject, were obtained from the Coriell Institute for Medical Research and cultured in DMEM supplemented with 20% FBS, 1% glutamine and 1% penicillin/streptomycin at 37°C. Phoenix-Ampho cells were purchased from ATCC (Manassas, VA, USA) and cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37°C. For serum starvation, cells were washed three times with pre-warmed DMEM medium and then incubated in DMEM medium for 18 hours. In some experiments, 50 μM hydroxychloroquine (CQ, Sigma) was present in serum-deprived and control cells to block the fusion of lysosomes with autophagosomes. For amino acid and serum starvation, cells were washed three times with pre-warmed Hank's buffered saline solution (HBSS) and then incubated in HBSS for 1 hour. After starvation, cells were re-supplemented with complete medium for 1 hour before harvest.

The ES-2 ovarian cancer cell line was labeled with luciferase for in vivo imaging. Conditioned media from Phoenix-AMPHO cells (ATCC) transfected with the retroviral expression plasmid pMSCV-Luciferase PGK-hygro using Lipofectamine 2000 (ThermoFisher, Waltham, MA) were centrifuged and passed through a pre-wet 0.45 μm filter. ES-2 cells were transduced by application of cleared retroviral supernatants mixed with 6 μg/mL Polybrene (AmericanBio, Natick, MA). A cell line stably expressing luciferase was selected by hygromycin and used for transfection experiments. Luciferase activity was assayed using 250 μg/mL D-luciferin (Perkin-Elmer, Waltham, MA) and the detection of luminescence with a Berthold Technologies Centro LB-960 plate reader and was normalized for cell number.

Validated and certified primary Normal Human Dermal Fibroblasts (NHDFs) isolated from human male neonatal foreskin were purchased from Lonza (CC-2509). HEK-293-T, HEK-293-A, VERO and BSC-40 cells were from Dr. Ian Mohr, NYU. Phoenix-Ampho cells were purchased from ATCC. All cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Fisher Scientific) supplemented with 5% Fetal Bovine Serum (FBS), 2 mM L-Glutamine and penicillin-streptomycin and maintained at 37°C, 5% CO₂. Where indicated, confluent cultures of NHDFs were growth-arrested by washing three times in PBS before being maintained in DMEM supplemented with 0.2% Fetal Bovine Serum (FBS), 2 mM L-Glutamine and penicillin-streptomycin for 72 h.

Retroviruses encoding rtTA or MEK1 genes were packaged in Phoenix-AMPHO cells obtained from ATCC. The supernatant-containing virus was filtered with 0.45 μM PVDF membrane. The target cells were infected with virus for 8 hrs. 48 hrs later, cells were selected in medium containing Puromycin (2 μg/ml) or Hygromycin (100 μg/ml) for 3 days. The positive infected cell populations were further sorted using GFP as a marker after overnight exposure to 1μg/ml doxycycline. GFP positive cells were then cultured and expanded in medium with doxycycline along with antibiotics. For the conditional RAF knockout cells, the inducible expression cells were created in the absence of 4-Hydroxytamoxifen.