1. Volume 12, Issue 5, Pages 1151-1164 (November 2003)
Tumor Suppressor ARF Degrades B23, a Nucleolar Protein Involved in Ribosome Biogenesis and Cell Proliferation 
Koji Itahana, Krishna P. Bhat, Aiwen Jin, Yoko Itahana, David Hawke, Ryuji Kobayashi, Yanping Zhang 
Molecular Cell 
Volume 12, Issue 5, Pages (November 2003)
DOI: /S (03)

2. Figure 1 ARF Interacts with B23
(A) Mass spectrometry detection ARF binding proteins. Extracts of U2OS cells infected with indicated adenoviruses were immunoprecipitated with ARF antibodies and were resolved on a silver-stained gel. Peptide bands unique in ad-ARF-infected samples are subjected for mass spectrometry.
(B) The binding between ectopically expressed ARF and B23. U2OS cells were transfected with plasmids encoding myc-B23 and/or infected with indicated viruses. Cell extracts were immunoprecipitated with an anti-ARF antibody and Western blotting was performed with indicated antibodies.
(C) The binding of endogenous B23 and endogenous ARF in HeLa cells.
(D) The binding between endogenous ARF induced by E2F1 with endogenous B23 in normal fibroblasts. WI-38 cells and U2OS cells were infected with indicated viruses. Cell extracts were immunoprecipitated with either ARF (left panel) or B23 (right panel) antibodies.
(E) No effect for RNase A treatment on ARF-B23 complex formation. Extracts from U2OS cells transfected with plasmids encoding ARF and myc-B23 were immunoprecipitated with ARF antibodies. RNase A (200 μg/ml) was added throughout immunoprecipitation and IP.
(F) MDM2 or p53 do not interact with B23. Extracts from U2OS cells infected with indicated viruses were immunoprecipitated with indicated antibodies (upper panel). WI-38 cells were exposed to UV (25J/m2) for 24 hr and cell extracts were immunoprecipitated with indicated p53 antibodies. Untreated WI-38 cells and p53 negative Saos2 and H1299 cells served as a control. Note that a nonspecific band at the position of B23 was observed in samples immunoprecipitated with DO1 antibody.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 Binding Domains for ARF and B23
(A) Requirement of N-terminal of ARF for binding with B23. Extracts from U2OS cells transfected with plasmids encoding deletion mutants of myc-ARF were immunoprecipitated with myc antibodies and blotted as indicated.
(B) Requirement of N-terminal of B23 for binding with ARF. Extracts from U2OS cells transfected with plasmids encoding ARF and deletion mutants of myc-B23 were immunoprecipitated with ARF antibodies and blotted as indicated.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3 ARF Promotes Rapid Degradation of B23
(A and B) Downregulation of endogenous B23 by ectopically expressed ARF. U2OS cells were infected with indicated viruses. Protein or RNA was harvested at indicated times post infections and analyzed by Western blotting or Northern blotting, respectively.
(C) Half-life assay of endogenous B23. Saos2 cells were infected with adenoviruses expressing indicated proteins. Twenty-four hours after infection, cells were pulsed with [35S]-methionine for 2 hr and then chased for the indicated length of time. Cell lysates were immunoprecipitated with an antibody specific to B23. The resulting B23 immunoprecipitates were separated electrophoretically by SDS-PAGE and visualized by autoradiography. The amount of labeled B23 protein at each time point was quantified on a PhosphoImager and normalized relative to the amount of radio labeled B23 present in cells following the 0 hr chase and the results are plotted.
(D) Downregulation of stably overexpressed B23 by ARF. Stable U2OS clones expressing myc-B23 were infected with indicated adenoviruses for 2 days and extracts were analyzed by Western blotting.
(E) Downregulation of endogenous B23 but not C23 by ARF. U2OS cells were infected with indicated viruses for 2 days and cell extracts were analyzed by Western blotting.
(F) Downregulation of endogenous B23 by endogenous ARF induced by E2F1 in WI-38 cells. WI-38 and U2OS cells were infected with indicated viruses for 2 days and cell extracts were analyzed by Western blotting.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 ARF Degrades B23 in a p53- and MDM2-Independent Manner
(A) p53 or MDM2 do not induce B23 degradation. U2OS and Saos2 cells were infected with indicated viruses for 1 day. Protein extracts were analyzed by Western blotting.
(B) ARF but not p53 decreases nucleolar and nucleoplasmic B23. Saos2 cells were infected with indicated viruses for 2 days (panels 2 and 3) and double-immunostained with indicated antibodies. Nuclei were visualized by DAPI staining. ARF or p53 infected cells were indicated by arrows.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
ARF-Induced B23 Degradation Involves B23 Polyubiquitination and the 26S Proteasome Pathway but Not CRM1-Mediated Nuclear Export
(A) The 26S proteasome inhibitor MG132 prevents ARF-induced B23 degradation. U2OS cells were infected with indicated viruses for 2 days. Cells were treated with 20 μM of MG132 for 12 hr before harvesting. Protein extracts were analyzed by Western blotting.
(B) ARF promotes polyubiquitination of B23. H1299 cells were transfected with indicated plasmids for 3 days. In vivo ubiquitination assay was carried out as described in Experimental Procedures. His-tagged B23 was precipitated by nickel-charged resin beads. Polyubiquitinated bands of His-B23 modified by HA-polyubiqutine were revealed using the HA antibody by Western blotting (upper panel). The membrane was reprobed with His antibody to reveal precipitated, nonubiquitinated His-B23 (middle panel). Cell extracts were analyzed by Western blotting using ARF antibody (lower panel)
(C) No effect of LMB on B23 degradation by ARF. U2OS cells were infected with indicated viruses for 2 days. Cells were treated with 10 μM of LMB for 12 hr before harvesting. Protein extracts were analyzed by Western blotting.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6 Downregulation of B23 Inhibits Ribosome Biogenesis
(A) A diagram of RNA processing (Ruggero and Pandolfi, 2003).
(B) Downregulation of B23 by siRNA and ARF. Saos2 cells were transfected with either a control scrambled RNA duplex (Scr) or B23 siRNA (B23) for 3 days, or infected with indicated viruses for 2 days. Cell extracts were analyzed by Western blotting.
(C and D) Blocking of 28S rRNA maturation by ARF overexpression and by B23 inhibition. Saos2 cells were either transfected with siRNA for 3 days or infected with indicated viruses for 2 days, pulse labeled with L-[methyl-3H]-methionine, and chased for the indicated times. An equal amount of radioactivity was loaded into each lane. Ethidium bromide staining of 28S rRNA is shown at the bottom. The ratio of densitometry signals of 28S/32S for lanes 3, 6, 9, and 12 calculated on a PhosphoImager are shown.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
The Effect of B23 Level on Cell Proliferation and Survival and a Role of ARF in Preventing Induction of B23 by Oncogenic Insult
(A) B23 induces cell cycle arrest in WI-38 cells but promotes S phase entry in Saos2 and p53−/− MEFs. WI-38, Saos2, and p53−/− MEF cells were infected with indicated viruses. Cells were harvested after 2 days of infection, stained with propidium iodide (PI), and the cell cycle distribution was determined by flow cytometry. Percentage of cells in the S phase is indicated.
(B) Cell death caused by B23 downregulation. WI-38 and U2OS cells were transfected with indicated siRNA for 3days and analyzed by flow cytometry as in (A). Percentage of cells in the sub-G1-phase is indicated. Cell extracts from cells transfected with scrambled siRNA (siScr) and B23 siRNA (siB23) were analyzed by Western blotting and the results are shown in the right panels.
(C) Inverse correlation between the level of B23 and ARF in MEFs during passage. Wild-type MEFs were serially passaged and cell extracts were isolated and analyzed by Western blotting.
(D) Levels of B23 in MEFs. Cell extracts from wild-type (P2) and ARF−/− MEFs (P4) were analyzed by Western blotting.
(E) H-Ras induce B23 in ARF−/− MEFs. Early (P4) and late (>P20) passage ARF−/− MEFs were infected with retroviruses carrying pBabe vector (vector) or pBabe-Ha-RAS (V12) (Ras). After 2 days of infection, cells were selected by puromycine (2 μg/ml) for 3 days and cell extracts were harvested and analyzed by Western blotting.
(F) H-Ras does not induce B23 in p53−/− and p53−/−/MDM2−/− MEFs. Indicated cells were infected and selected as in (E). Cell extracts were harvested and analyzed by Western blotting.
Molecular Cell  , DOI: ( /S (03) )