Pmko 1 puro gfp shrna

The pRetrosuper Myc shRNA and pMKO.1‐puro GFP shRNA were gifts from Addgene (plasmids #15662 and #10675, respectively; Cambridge, MA, USA) 33, 34. To obtain cells stably expressing decreased levels of MYC, the MYC shRNA vector (AMU‐ML2/MYCsh) or the control GFP shRNA vector (AMU‐ML2/GFPsh) was introduced into AMU‐ML2 cells. The retroviral plasmids were packaged into 293T cells using the pCL10A vector. Viral supernatants were harvested 96 h after transfection and filtered before infection. The cells were infected with retroviruses in the presence of 8 μg·mL⁻¹ Polybrene (Sigma‐Aldrich). Antibiotic selection (puromycin; 0.3 μg·mL⁻¹; Wako) was begun 48 h after infection and continued for at least 3 days. Following infection and antibiotic selection, the cells were examined for MYC protein levels using western blotting.

OCI-AML3 cells [15 (link)] (human leukemia cell line kindly provided by Dr. Mark Minden, Ontario Cancer Center, Canada) and OCI-AML–3 cells stably expressing shRNA targeting p53 and vector control [16 (link)] (kind gifts from Dr. Paul Corn, University of Texas MD Anderson Cancer Center), mouse embryonic fibroblasts (MEF) ^wt/wt, ^{AMPK -/-} [17 (link)] (kind gifts from Dr. Juan Fueyo-Margareto, Neuro-oncology, MD Anderson Cancer Center) were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum (FCS). HL60, HEK-293T and REH cells were obtained from the ATCC (Manassas, VA). Phoenix Amphotrophic retrovirus packaging cells were obtained from Orbigen (San Diego, CA). A set of 7 shRNAmirs each targeting BECLIN 1 plus non-silencing control lentiviral vector were obtained from Open Biosystems (Huntsville, AL). Lentiviral packaging plasmids MD2.G (plasmid 12259) and psPAX2 (plasmid 12260), both constructed by the laboratory of Didier Trono, plus retroviral vectors pMKO.1 puro p53 shRNA#2 (plasmid 10672), pMKO.1 puro GFPshRNA (plasmid 10675), and pBABE-puro mCherry-EGFP-LC3B (plasmid 22418) (constructed in the laboratory of Jayanta Debnath) were obtained from Addgene (Cambridge, MA).