1. Volume 45, Issue 1, Pages 123-131 (January 2012)
DNA Damage Signaling Triggers Degradation of Histone Methyltransferases through APC/CCdh1 in Senescent Cells 
Akiko Takahashi, Yoshinori Imai, Kimi Yamakoshi, Shinji Kuninaka, Naoko Ohtani, Shin Yoshimoto, Satoshi Hori, Makoto Tachibana, Emma Anderton, Takashi Takeuchi, Yoichi Shinkai, Gordon Peters, Hideyuki Saya, Eiji Hara 
Molecular Cell 
Volume 45, Issue 1, Pages (January 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2012 45, 123-131DOI: (10.1016/j.molcel.2011.10.018)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Degradation of G9a/GLP in Senescent Cells
(A) IMR-90-ER:Ras cells cultured with OHT for indicated days were subjected to western blot analysis using antibodies shown left (, phospho-specific antibody) or to the analysis of intracellular ROS levels.
(B and C) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 10 days were subjected to the analysis of senescence-associated β-galactosidase (SA-β-gal) activity (B) or H3K9me1/2 level after histone extraction (C).
(D) Early passage (40PDL) TIG-3 cells infected with retrovirus encoding p16INK4a or empty vector for 4 days were subjected to western blotting for indicated proteins or to the analysis of BrdU incorporation, or immunofluorescence staining for γH2AX foci.
(E) IMR-90-ER: Ras cells were cultured for 10 days with (+) or without (−) OHT. Cells were then incubated with (+) or without (−) 10 μM of MG132 for 12 hr and were subjected to western blotting for indicated proteins.
(F) Early passage TIG-3 cells infected with retrovirus encoding HA-tagged ubiquitin were subsequently infected with retrovirus encoding oncogenic Ras (+) or empty vector (−). Cells were then subjected to transfection with indicated previously validated siRNA oligos and were treated with 10 μM of MG132 for 12 hr and then subjected to western blotting for indicated proteins before (lanes 1 and 2) or after (lanes 3–14) immunoprecipitation with antibodies shown top (IP). The error bars in all panels represent the means ± SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2 APC/CCdh1 Promotes Degradation of G9a/GLP
(A) IMR-90-ER:Ras cells cultured for 9 days with (+) or without (−) OHT were incubated with 1 mM of caffeine and 10 mM of NAC or control (H2O) for another 24 hr and were subjected to western blotting using antibodies shown right, or to analysis of intracellular ROS levels. (B and D) IMR-90-ER:Ras cells cultured for 6 days with (+) or without (−) OHT were subjected to transfection with indicated siRNA oligos three times (at 2 day intervals) and were subjected to western blotting using antibodies shown right before or after histone extraction (Histone). (C) Early passage TIG-3 cells treated with (+) or without (−) 0.5 μg/ml of doxorubicin (DXR) for 12 hr were subsequently treated with (+) or without (−) 10 μM of MG132 for 10 hr. Cells were then subjected to western blotting using antibodies shown right. (E and F) Early passage TIG-3 cells infected with retrovirus encoding Cdh1 or empty vector were subjected to western blotting for indicated proteins before or after histone extraction (E) or to SA-β-gal analysis (F). (G) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 10 days were subjected to western blotting for indicated proteins before (Input) or after immunoprecipitation with indicated antibodies (IP). (H) IMR-90-ER:Ras cells cultured with OHT for 6 days were subjected to indicated siRNA transfection three times (at 2 day intervals) and were subjected to western blotting for indicated proteins before (Input) or after immunoprecipitation with indicated antibodies (IP). (I) IMR-90-ER:Ras cells cultured for 10 days with (+) or without (−) OHT were treated with 10 μM of MG132 for 12 hr and were subjected to western blotting for indicated proteins before (Input) or after immunoprecipitation with indicated antibodies (IP). Cyclin B and Vinculin were used as positive and negative control, respectively. The error bars in all panels represent the means ± SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Modality of Substrate Recognition and Mechanism of APC/Ccdh1 Activation in Senescent Cells
(A and B) Flag-tagged full length wild-type, D box point mutants, or truncation mutant of G9a (A) or GLP (B) was ectopically expressed with (+) or without (−) Myc-tagged Cdh1 in HEK293T cells. Cells were then treated with (+) or without (−) 10 μM of MG132 for 12 hr and subjected to western blotting for indicated proteins.
(C and D) Flag-tagged full-length or truncation mutant of G9a (C) or GLP (D) was cotransfected with indicated siRNA oligos in HEK293T cells expressing HA-tagged ubiquitin. Cells were then treated with MG132 for 12 hr and subjected to western blotting for indicated proteins before (Lysate) or after immunoprecipitation with anti-Flag antibody (IP).
(E) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 10 days were trasnfected with a expression vector encoding GFP-Cdc14B fusion protein. Micrographs of representative cells were shown.
(F and G) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 6 days were subjected to transfection with indicated siRNA oligos three times (at 2 days interval) and were subjected to western blotting for indicated proteins or to RT-qPCR. The error bars in all panels represent the means ± SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 APC/CCdh1 Regulates SASP
(A and B) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 10 days were subjected to analysis of RT-qPCR for indicated genes or to ChIP analysis using the antibodies shown top.
(C) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 6 days were subjected to transfection with indicated siRNA oligos three times (at 2 day intervals) and were then subjected to RT-qPCR for indicated genes.
(D and E) IMR-90-ER:Ras cells cultured with (+) or without (−) OHT for 6 days were subjected to transfection with indicated siRNA oligos three times (at 2 day intervals) and were then subjected to ChIP analysis using the antibodies shown top.
(F) Early passage TIG-3 cells infected with retrovirus encoding G9a, GLP, or Vector were infected with retrovirus encoding oncogenic Ras (V12) or empty vector (Control) for 10 days and were subjected to western blotting for indicated proteins or to RT-qPCR analysis for indicated gene expression.
(G) A model of H3K9me2 regulation in Ras-induced senescence. DNA damage activates APC/CCdh1 through p21CIP1 and Cdc14B-dependent pathways. DDR also reduces DNMT1 level which, in turn, provokes DNA damage, thereby establishing a positive feedback loop to sustain DDR. Oncogenic Ras signaling therefore establishes an autonomous APC/CCdh1 activation, leading to degradation of G9a/GLP and resulting epigenetic alteration. The error bars in all panels represent the means ± SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions