Supplementary Figure 1: HeLa and H1299 cells exhibit signs of DDR activation and detectable levels of ARF expression, but Chk2 silencing does not affect p14^ARF protein levels.

a. Immunoblot (IB) analysis shows decreased phospho-Chk2 levels upon ATM inhibition (ATMi) in H1299 and HeLa cells, demonstrating the effectiveness of the inhibitor. b. IBs depicting signs of DDR activity, assessed by gH2AX (see also Fig. 1a), and detectable levels of p14^ARF expression in HeLa and H1299 cells. c. IBs demonstrating that Chk2 silencing in HeLa and H1299 cells has no effect on p14^ARF levels. p14^ARF mRNA levels are induced by oncogenic stimuli (Cdk4, Ras and E2F1), but are unaltered after inhibition or silencing of ATM. Bars represent quantification of p14^ARF mRNA levels as assessed by semi-quantitative real time RT–PCR in: (d) Human Bronchial Epithelial Cells (HBECS) (KT: hTERT, Cdk4 and KT transfected with activated K-Ras^V12), NARF cells (with or without IPTG induction) and Saos2-E2F1-ER (non-induced, TAM-induced and TAM-induced + ATMi), and in: (e) H1299, HeLa, HBECs cells transfected with control siRNA, siATM, DMSO (control) and Ku55933 (ATMi), induced NARF2 treated with Doxorubicin and Doxorubicin +Ku55933, respectively. (p<0.005, t-test, error bars indicate SDs, n = 4 real time RT–PCR runs). Silencing of ATM enhances oncogene-induced p14^ARF expression in BJ cells and HBECs. f. IB analysis complementing Fig. 1c results showing the status of DDR activation and p14^ARF levels in immortalized HBECs (hTERT, Cdk4) and immortalized HBECs with K-Ras^V12. Genetically manipulated HBECs expressing various oncogenes^16,17,76 were employed as normal cells do not demonstrate signs of DDR activation⁴⁷ and ARF levels are negligible¹. g. IB analysis results showing that β-catenin transfected diploid BJ human fibroblasts exhibit DDR activation, while upregulation of p14^ARF levels require additionally low serum conditions, as previously reported²⁰. h. IB analyses, complementing panel g, demonstrating that the increased p14^ARF protein levels in BJ primary human fibroblasts due to β-catenin transfection and serum depletion was further enhanced after silencing of ATM. ATM regulates p14^ARF protein stability. i Silencing of ATM (shATM H1299 cells) protects ARF from DNA damage mediated downregulation. Oncogenic stimuli compete with active ATM in regulating ARF expression. j. IB analysis of p14^ARF in H1299 cells infected with pBabe (control) or pBabe-Ha-Ras^V12 and in the presence or absence of ATM inhibitor Ku55933, showing that the oncogenic challenge of H1299 cell (that already harbour mutant and activated K-Ras) decreases the endogenous levels of ARF that were re-established when ATM was inhibited. Apparently, the oncogene-ARF pathway, in this setting, has reached an activation plateau and any additional oncogenic stimulus activates ATM leading to ARF suppression. b. Two signalling routes lead to ARF induction, oncogenic challenge¹ and ATM suppression. Given that oncogenes activate ATM, as well^4,7,47,50,60, oncogenic insults trigger two pathways with opposing effects on ARF expression. The outcome of this antagonism will depend on whether the rate of ARF production by oncogenes exceeds or not the rate of ARF destruction by the oncogene induced ATM pathway. Actin serves as loading control. ATMi = Ku55933 addition, PBGD = Porphobilinogen deaminase (house-keeping gene), Dox = Doxorubicin, ATMi = Ku55933 addition, ctrsi = control siRNA, TAM = 4-OH-Tamoxifen.