Cell Culture and Treatment
The RKO, U2OS, H460, 293FT, HCT116, and H1299 cell lines were obtained from ATCC and cultured in DMEM medium with 10% fetal bovine serum supplement and 1% penicillin-streptomycin. The ATG5+/+ and ATG5-/- MEFs were a kind gift from Dr. Mizushima (Tokyo Medical and Dental University) and cultured in DMEM medium with 10% fetal bovine serum . The MDA-MB-231 was also obtained from ATCC and cultured in McCoy's 5A medium. The Rh30 cell line was kindly given by Peter Houghton (St. Jude Children's Research Hospital) and cultured in RPMI medium with 10% fetal bovine serum. Normal human epidermal keratinocytes (NHEKs) were obtained from the Vanderbilt Skin Disease Research Core and cultured as previously described . Primary human mammary epithelial cells (HMECs) were purified from normal breast tissue obtained by the Vanderbilt-Ingram Cancer Center Human Tissue Acquisition and Pathology Shared Resource Core, and were isolated and grown as previously described [22, 60].
The following chemotherapeutics were used in treatment of cell lines mentioned above as described in results 8 Gy 137Cs ionizing radiation, 0.13 mM 5-FU (APP Pharmaceuticals), 20 μM etoposide (Bedford Laboratories), 5 μg/mL cisplatin (APP Pharmaceuticals), 5 nM paclitaxel (Sigma), 40 nM rapamycin (Calbiochem). Lysosomal inhibitors were used at final concentration of 10 μg/mL of E64d (Calbiochem 330005) and pepstatin A (MP Biomedical 195368).
Cell Transfection and Small Interfering RNA
The following targeting sense strand sequences were used for siRNA: Dharmacon siControl (Non-Targeting siRNA #1) UAGCGACUAAACACUCAA; Dharmacon siISG20L1-1 CAGCAAGGUUCACGGAUAUUU; siISG20L1-2, AUACUAAGCAAGCGAGGGAUU; siISG20L1-3, CUCAAUUGGAAACGUGAAAUU. Dharmacon siRNA ISG20L1 pools consisted of the above targeting vectors plus siISG20L1-4 CAGCAGGGCCACUCGUCUA. Dharmacon siRNAs were reverse transfected into H460, U2OS, and RKO cells (4.5 × 105) with Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol.
To knockdown p53 in NHEK cells, a 19-bp short hairpin RNA, corresponding to nucleotides 611 to 629 of p53 RNA (GenBank NM000546), was annealed and cloned into the self-inactivating lentiviral vector (H1-LV) that contains a GFP reporter gene under control of human ubiquitin C promoter for monitoring infection efficiency. A scrambled oligonucleotide was designed as a negative control and also cloned in the H1-LV vector. These lentiviral vectors were transfected using CaPO4 methods into 293FT cells. After 48 h viral medium was harvested and with the addition of 8 μg/mL polybrene used to infect NHEK cells.
293FT cells were transfected using Fugene 6 (Roche) to make pSico lentivirus. To knockdown p73 in MDA-MB-231 and Rh30, cells were infected with the pSico lentivirus system that expresses shRNA targeting all isoforms of p73 as previously described . Forty-eight h later, cells were treated with rapamycin (40 nM) and RNA harvested 24 h later.
293FT cells were transfected using Lipofectamine2000 with either pCEP4 empty control or cDNAs encoding p53, TAp63γ, TAp73β, or ΔNp63α and harvested 24 h later for RT-PCR or Western analysis.
Clonogenic Survival Assays were performed in HCT116, RKO, H1299 cells, as well as ATG5+/+ and ATG5-/- MEFs transformed with SV40 large T antigen obtained from Dr. Mizushima . For all cell lines, Lipofectamine2000 was used to transfect either pCEP4 empty vector control or ISG20L1 in 60 mm dishes. Twenty-four h after transfection, cells were selected for 10 days under the appropriate hygromycin B concentration determined per cell line. Colonies were Wright stained and analyzed using the Biorad Quantity One software.
Western Analysis and Antibodies
Western analyses were performed as previously described . Fourteen percent SDS-polyacrylamide gels were used for analysis of LC3 using anti-MAP1LC3-II (Abgent AP1802a). Additional antibodies used for protein detection: anti-p53 (Santa Cruz Biotechnology, PAb1801), anti-β-Actin (Sigma-Aldrich, A5441-0.2 mL), anti-PARP (Cell Signaling, #9542), anti-Caspase-3 (Cell Signaling, #9662), anti-p73 (Bethyl A300), p63 (4A4) (Santa Cruz, sc-8431), and anti-ISG20L1 (Bethyl Laboratories, rabbit affinity purified antibody). A peptide for ISG20L1 antibody production was designed at the C-terminus of ISG20L1, outside of the functional exonuclease domain found from amino acids 111-275, with the intent to increase antigenicity and accessibility of the antibody while decreasing possible cross-reactivity. The peptide product sequence "HGSRGGAREAQDRRN" targets amino acids 311-325 of ISG20L1 and these 15 amino acids are unique to the ISG20L1 sequence.
RNA Isolation and Real-Time Analysis
RNA isolation and all subsequent quantitative real-time PCR (qRT-PCR) analyses were performed as described previously . All primer sets were run under the following cycling conditions: 95°C for 3 minutes followed by 40 cycles of: 95°C for 10 sec and annealing at 60°C for 45 sec, with data acquisition during each cycle. Melting curve analysis following PCR cycling was used to determine purity and quality of PCR product.
Immunofluorescence, Immunohistochemistry, and Electron Microscopy
For immunofluorescence analysis, cells were grown on glass coverslips and fixed in a 4% paraformaldehyde solution for 10 min at room temperature. After rinsing with PBS, the cells were permeabilized with 0.5% Triton X-100 for 10 min. Following another rinse with PBS, cells were blocked for 15 min at room temperature with 5% BSA-PBS solution. The ISG20L1 (Bethyl) and FLAG antibodies (Sigma, F3165 anti-FLAG M2) were diluted in 1% BSA-PBS and incubated on cells at 37°C with 5% CO2 for 1 h. The coverslips were washed 3× with PBS and placed in 2° rabbit anti-Alexa Flour 546 and mouse anti-Alexa Flour 488, respectively for 1 h at room temperature, in the dark. The cells were washed 3× with PBS and counterstained with DAPI. All images were obtained using 1000× magnification on a Zeiss Axioplan microscope equipped with a Zeiss camera and software.
Direct immunofluorescence was performed on U2OS cells stably expressing mRFP-GFP-LC3. The mRFP-GFP-LC3 expression vector was kindly provided by Dr. Yoshimori (Osaka University)  and Dr. Mizushima (Tokyo Medical and Dental University) . U2OS stably expressing the tagged LC3 protein were generated by transfecting the cells with the mRFP-GFP-LC3 expression vector using FuGENE 6 (Roche, Indianapolis, IN) and selecting in geneticin (Cellgro, Manassas, VA). Engineered U2OS cells were then transfected with either pCEP4 control or ISG20L1 expression plasmids and treated for 24 h with 5-FU. The cells were fixed and analyzed as above using a Zeiss Axioplan. Fifty cells were counted, without knowledge of the plasmids expressed, and RFP-only foci are reported as a percentage of total foci.
For immunohistochemistry analysis, cells were grown on glass coverslips. The cells were fixed, and permeabilized as indicated above for IF analysis. Washes were done in 1× TBS/0.1% Tween-20 (1× TBST), and cells were blocked overnight rocking at 4°C in 5% normal goat serum diluted in TBST. The coverslips were stained specifically for the cleaved LC3 using the Abgent LC3 specific 1° antibody (Abgent AP1806a) for 30 min at room temperature. The coverslips were then washed 3 times in TBST. The secondary used was the Dako Cytomation LSAb2 system HRP kit (K0673) according to manufacturer's protocol. Cells were analyzed for LC3 staining and counted at 200× magnification.
U2OS cells were reverse transfected using Lipofectamine2000 with Dharmacon Nonsilencing control or siRNA targeting ISG20L1. Three days after reverse transfection, cells were treated or not for 24 h with 5-FU to induce autophagy. Cells were harvested, washed with PBS, and exposed to 2% glutaraldehyde for fixation. Sample were rinsed in buffer, postfixed in 1% OsO4 for 1 h, dehydrated through an ethanol series and transferred into Epon resin. Ultrathin sections (60-70 nm, silver-gray) were obtained using a Reichert Ultracut E microtome with a diamond knife, transferred to formvar-coated grids, and examined on a Phillips CM-10 transmission electron microscope (FEI, Hillsboro, OR), operating at 80 kV, and images were captured with an AMT 2 mega pixel camera (Advanced Microscopy Techniques, Danvers, MA).
Two replicates were performed and each time 25 micrographs were counted blindly for each control and ISG20L1 knockdown. Additionally, cells were photographed in an un-biased fashion according to their placement on the grid. Images were quantified using ImageJ software and taking into account various acceptable methods [39, 42]. We set to scale the pixel ratio to microns and used measurement analysis to quantify the area occupied by autophagosome and autolysosomes as compared to the total cytoplasmic area excluding the nucleus. Autophagosomes were defined as double or multiple membrane structures surrounding cytoplasmic material, and autolysosomes were defined as single membrane structures surrounding cytoplasmic constituents at various levels of degradation .
Flow Cytometric Analyses
Flow cytometry was performed as previously described using a FACSCaliber instrument (Becton-Dickinson) . Annexin V-FITC staining detected by flow cytometry was performed using the Annexin V-FITC apoptosis detection kit (BD Pharmingen, 556547).
Chromatin Immunoprecipitation Analyses
HMECs were treated or not with 10 ug/mL cisplatin for 24 h and chromatin was prepared as previously described . PCR amplification was performed using primers ISG20L1 forward CAGCCTGTCCAACATGGC and ISG20L1 reverse GCTGAGGCCATAACTTGGAAA, GAPDH forward CACCAGCCATCCTGTCCTCC and GAPDH reverse GTTCCTTCCCAGCCCCCACT, and p21 forward GCTTGGGCAGCAGGCTG and p21 reverse AGCCCTGTCGCAAGGATC as previously described . PCR was performed using one cycle of 5 min at 95°C; followed by different number of cycles as indicated below of: 95° for 30 s, annealing temperature as indicated below for 45 s, and 30 sec of 72°C; to be finished with 10 min at 72°C. AEN 40 Cycles Anneal 54°C, GAPDH 35 Cycles Anneal 62°C, and p21 35 Cycles Anneal 57°C. Amplified DNA was resolved on a 6% polyacrylamide gel and stained after with ethidium bromide.
To attain sufficient levels of p73 for ChIP analysis, ~1.7 × 107rapidly growing Rh30 cells were treated for 24 h using vehicle control or 40 nM rapamycin. The samples were prepared and Genpathway analysis performed as previously described  using the p73 antibody (Bethyl Laboratories, A300) for immunoprecipitation.
Cells were counted and 2 × 106 cells were removed and washed in PBS for DNA laddering analysis. Procedure was followed according to the Roche Apoptotic DNA-Ladder Kit (11 835 246 001). In brief, cells were lysed in an equal volume of proprietary lysis buffer, incubated for 10 min at room temperature, 100 μl of isopropanol was added and vortexed prior to loading the sample onto filter tubes. Filter tubes were spun 2× 1 min at 8000 rpm and washed after each spin with 500 μl washing buffer. After discarding flow through, filter tube samples were placed in collection tubes and 100 μl elution buffer was added and then spun for 1 min at 8000 rpm. DNA obtained from samples was run on a 1% agarose gel next to 1 kb DNA ladder and positive control DNA (U937 cells treated with camptothecin) supplied from Roche.
Data were analyzed where indicated using the Student's t test for statistical significance. P values are indicated in the figure legends and text. Standard deviation and error were calculated and represented in bar graphs where indicated.