(A) Western analysis of p53-null MEFS (high ARF) and MEFs from the p53/ARF double knockout mouse (p53/ARF DKO) following infection with short hairpin to silence Beclin-1 (shBec1) for the levels of actin (loading control), ARF and Beclin-1.
(B) Bioluminescent imaging of xenograft tumors derived from early passage MEFs from the p53 −/− mouse, or p53/ARF double knockout (DKO) stably-infected with luciferase virus, and then infected with the retroviruses indicated (shARF or shBec) for 48 hours. Tumors were imaged after 21 days; equivalent results were seen after 30 days, and all measurements were consistent with caliper measurements.
(C) Analysis of the combined imaging data from two independent experiments depicted in B, using three mice for each sample per experiment (6 samples total per retrovirus combination); all values represent the raw mean value of bioluminescence (mean BLI) along with standard error of the mean (SEM).

(A) Western analysis of whole cell lysate from p53 −/− MEFs (passage 3) untreated or incubated in Hank’s Buffered Salt Solution (HBSS) for the indicated timepoints (starvation) using antisera to actin (loading control) and ARF.
(B) Western analysis of MEFs with wt p53 (passage 2), untreated or incubated in Hank’s Buffered Salt Solution (HBSS) for the indicated timepoints (starvation) using antisera to actin (loading control) and ARF.
(C) Analysis of the half life of ARF in untreated and nutrient-deprived cells. p53-null MEFs were untreated (UNT) or incubated for 2 h in HBSS, followed by supplementation with complete media containing 40 ug/uL cycloheximide to halt new protein synthesis. Cells were harvested and analyzed by western blotting for ARF at the indicated timepoints; the data depicted are representative of two independent experiments.
(D) Immuno-electron microscopy using ARF antisera followed by Protein G-Gold in mouse embryo fibroblasts from the p53-knockout mouse following two hours treatment with HBSS (starvation). Arrows point to gold particles in mitochondria and nucleoli. Scale bar is 500 nm.

(A) Western analysis for LC3 II in p53 −/− MEFs following nutrient starvation (HBSS). Cells were infected for 48 hours with a short hairpin for p53 (shp53, negative control) or short hairpin for ARF (shARF), and incubated for 2 hrs in HBSS. Data depicted are representative of three independent experiments; the depicted data were obtained from a single blot that was cropped for optimal data presentation. The difference in LC3 II in unstressed cells in lanes 1 and 3 was not consistent or reproducible from experiment to experiment.
(B) Electron microscopy of p53-null MEFs infected with shp53 (negative control, top panels) or shARF (bottom panels) following 2 hour incubation in HBSS (starvation). Scale bars: Left panels, 5 um. Right panels 500 nm.
(C) The degradation of long-lived proteins in p53-null MEFs infected with short hairpin to p53 (negative control, black line) is increased compared to those infected with short hairpin to ARF (shARF, gray line) following starvation in HBSS for 24 hours; results are normalized to cell number. The x axis indicates timepoints of sampling. Data are representative of two independent experiments.
(D) The viability of p53-null cells infected with short hairpin to p53 (MEF p53 −/−) or shARF following 24 hour treatment with HBSS is depicted; equal numbers of viable cells were plated at the 0 timepoint (1 × 10⁶), and cell viability was determined using the ViaCount assay and the Guava Personal Cell Analysis machine. Data depicted are the averaged results from three independent experiments plus standard error.

(A) Bioluminescent imaging of tail-vein injected, luciferase-expressing B2998 B-cell lymphoma cells from the Eμ-myc mouse, infected with vector control or two different short hairpins for ARF, shARF157 or shARF56. Most tumor cells track to the lymph nodes and meninges. Results are at day 7 following tail vein injection; equivalent results were obtained at day 14. Data depicted are representative of two independent experiments, using 3 mice for each retrovirus (shControl, shARF157, shARF56). The middle panel depicts western analysis for p16, ARF and actin control in infected lymphoma cells from the B2998 lymphoma, 48 hours after retroviral infection. The right panel depicts the combined data from 2 independent experiments using 3 mice per virus, along with standard error of the mean. Silencing of ARF, but not infection with short hairpin control vector, reduces tumor volume by 60% (p<0.01).
(B) The viability of B2998 cells with silenced ARF is decreased following HBSS treatment for 24 hours. Cell viability was determined using the ViaCount assay and the Guava Personal Cell Analysis machine. Data depicted are the averaged results from three independent experiments plus standard error. The p value reflects comparison between the second and fourth column.
(C) Silencing ARF in B2998 lymphoma cells reduces the steady state level of autophagy. Western analysis for ARF, actin and p62^SQSTM1 (which is degraded by autophagy) in cells infected with shControl or each of two ARF short hairpins (shARF157 and shARF56).
(D) Silencing ARF in a primary T cell lymphoma from the p53 −/− mouse impedes tumor progression. Results are at day 2 following tail vein injection; equivalent results were obtained at day 4. Results are representative of two independent experiments. The right panel depicts western analysis for ARF and loading control (actin).