Mission shrna bacterial stocks for atg5

H460 cells were obtained from ATCC® (NCI-H460; Gaithersburg, MD). p53 knockout H460 cells (H460crp53) were generated as described elsewhere (36 ). Briefly, cells were co-transfected (3 × 10⁶ cells in 10-cm dish) with 1 μg CRISPR-Cas9 plasmid targeting the p53 loci and 1 μg of a homology-directed repair plasmid for p53 (both from Santa Cruz Biotechnology® Inc., Santa Cruz, CA). Cells were transfected using PolyJet™ reagent (SignaGen Laboratories, Rockville, MD) following the manufacturer’s guidelines. After 72 h, cells were exposed to 2.5 μg/ml puromycin with daily media exchanges to replenish the selection agent. After all cells transfected with 1 μg of a control CRISPR/Cas9 plasmid (Santa Cruz Biotechnology, Inc.) were killed (~96 h), the cells were allowed to recover and grow as individual colonies, which were then selected and examined for expression of p53 by Western blotting. shATG5 H460 cells were generated as described below. Mission shRNA bacterial stocks for ATG5 were purchased from Sigma-Aldrich (St. Louis, MO). Lentiviruses were produced in HEK 293TN cells co-transfected using EndoFectin™ Lenti Transfection Reagent (GeneCopoeia, Rockville, MD) with a packaging mixture of psPAX2 and pMD2.G constructs (Addgene, Cambridge, MA). Media containing the viruses was used to infect the H460 cells.

H460 NSCLC cells were obtained from ATCC (NCI-H460). p53 knockout H460
were generated by co-transfecting cells (3×10⁶ in 10 cm dish)
with 1 μg CRISPR-Cas9 plasmids targeting the p53 loci (Santa Cruz
Biotechnologies; cat #sc-416469) and 1 μg a homology directed repair
plasmid (Santa Cruz, cat. #sc-416469-HDR) expressing a puromycin selection
marker. Cells were transfected using PolyJet reagent (Signagen) following the
manufacturer’s guidelines. After 72 hr, cells were exposed to 2.5
μg/mL puromycin with daily media exchanges to replenish the selection
agent. After all cells in control plates were killed (96 h), puromycin was
removed and the cells allowed to recover and grow as individual colonies, which
were then individually selected and examined for expression of p53. For the
generation of shATG5 H460 cells, Mission shRNA bacterial stocks for ATG5 were
purchased from Sigma Aldrich. Lentiviruses were produced in HEK 293TN cells
co-transfected using EndoFectin^™ Lenti Transfection Reagent
(GeneCopoeia, 1001–01) with a packaging mixture of psPAX2 and pMD2.G
constructs (Addgene). The media including viruses were used to infect H460
cells.