nature05268-s1.doc - Legends for Supplementary Figures Suppl Fig 1 Induction of senescence of BJ diploid fibroblasts by mos overexpression is dependent

nature05268-s1.doc - Legends for Supplementary Figures...

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Legends for Supplementary Figures Suppl. Fig. 1. Induction of senescence of BJ diploid fibroblasts by mos overexpression is dependent on activation of the DNA DSB checkpoint pathway. a, Immunoblot analysis of BJ diploid fibroblasts non-infected (-) or infected with empty vector ( pBabe ) or a retroviral vector expressing mos . b-c, Suppression of mos -induced senescence by ATM depletion. b, Images of SA- -gal-stained BJ cells transfected with no siRNA (no si), control siRNA (ctl si) or siRNA specific for atm ( atm si). Means and SD of the percentages of SA- -gal-positive cells are indicated. c, Means and SD of BrdU- positive BJ cells transfected with control siRNA or atm -specific siRNA. The latter consisted of a pool of 2 oligonucleotides of the following sequences: 5'-CUA ACA AAC AGG UGA UAU AUU-3', 5'- AAA GGC CCA AGC UCC UCC UUU-3' (Dharmacon, siGENOME). d, Depletion of ATM does not compromise activation of ERK by mos . ATRIP serves as a loading control. Suppl. Fig. 2. Demonstration that oncogene-induced senescence is dependent on activation of the DNA DSB checkpoint pathway using atm -specific siRNA with a different sequence than the one used in Fig. 1 and Suppl. Figs 1,3-5,7. SA- -gal ( a ) and BrdU ( b ) analysis of diploid fibroblasts expressing the indicated oncogenes and transfected with control siRNA (ctl si) or siRNA specific for atm ( atm si) that consisted of a pool of 3 oligonucleotides of the following sequences: 5'-AAU UGC UGA UAU AGG CAA AUG UUG C-3', 5'- AUA ACG UCU CGA AGA ACA AAG GCC C-3', 5'-AAU AGA AGA AGU AGC UAC ACU GCG C-3' (Invitrogen, ATM Stealth Select 3 RNAi). 1
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Suppl. Fig. 3. Induction of senescence of MRC5 diploid fibroblasts by cdc6 overexpression is dependent on activation of the DNA DSB checkpoint pathway. a, Immunoblot analysis of MRC5 diploid fibroblasts non-infected (-) or infected with empty vector ( pBabe ) or a retroviral vector expressing cdc6 . b-d, Suppression of cdc6 -induced senescence by ATM depletion. b, Images of SA- -gal-stained MRC5 cells transfected with control siRNA (ctl si) or siRNA specific for atm ( atm si). Means and SD of the percentages of SA- -gal-positive cells are indicated. c, Means and SD of BrdU-positive cells transfected with control or atm -specific siRNA. d, Efficiency of ATM depletion. ATRIP serves as a loading control. e-g, Suppression of cdc6 -induced senescence by p53 depletion. e, Images of SA- -gal-stained MRC5 cells infected with a control short hairpin retroviral vector (ctl sh) or a pSUPERretro vector expressing a short hairpin specific for p53 ( p53 sh; ref. 30). Means and SD of the percentages of SA- -gal- positive cells are indicated. f, Means and SD of BrdU-positive cells infected with control or p53 - specific short hairpin vectors. g, Efficiency of p53 depletion. Actin serves as a loading control.
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