Supplementary Figure 3: deCDKN1A contains key enhancer elements that regulate OIS in a p53-dependet fashion.

(a) The same cell extracts as in Figure 2g were blotted with an antibody against HRAS. (b) qRT-PCR analysis of CDKN1A mRNA levels performed with the indicated BJ-indRAS^G12V cell populations following induction of RAS^G12V. (n=3; *P<0.05, two-tailed Student’s t-test). (c) MCF-7 cells were transfected with the indicated reporter vectors, treated with Nutlin-3a 5-10 hours later, and harvested 25-30 hours later. The relative luciferase activities (Firefly/Renilla) were normalized to the Ctrl reaction. (d and e) The same cell populations as in b panel were subjected to BrdU labeling and β-gal assays to assess proliferation and OIS, respectively. N=2; for each condition at least 150 cells were count. *P<0.05, two-tailed Student’s t-test. (f) Western blot analysis of BJ-indRAS^G12V CDKN1A KO and control cells. P53 and HSP90 were used as controls.