Fig. S1. Analysis of BrdU incorporation in cells treated with LY
ARF-null, p53+/+ MEFs were plated at a density of 200,000 cells per 6 cm dish, on
glass coverslips. After overnight incubation, serum was washed off, and cells were
incubated with or without 5% FCS. Following 4 hours in the presence or absence of
serum, cells were pulsed with BrdU for an additional 2 hours, and then fixed and
stained for BrdU. BrdU positive nuclei were visualized by fluorescence microscopy,
as described earlier [18]. Bars represent average and standard deviation of the
percentage of BrdU positive nuclei.
Fig. S2. LY294002 inhibits cisplatin-induced p53 induction in wild type MEFs
Wild type MEFs were plated and treated as in figure 2f. p53 was detected with the
CM5 antibody. GAPDH was used as a loading control.
Fig. S3. Effect of LY294002 on p53-dependent transcriptional activation by
CisPt.
5
HCT116 cells were plated in 24-well culture dishes, at a density of 10 cells/well. 16
hours later, cells were transfected with a combination of PUMA-luciferase reporter
plasmid DNA (20ng/well) together with 500ng of expression plasmid DNA encoding
either p53-specific shRNA (p53 si RNA) or an inactive derivative thereof, containing
3 base-paired mutations within the region complementary to the p53 RNA sequence
(mutant si). Cells were harvested 48 hours later. 6 hours before harvest, LY294002
was added to a final concentration of 20M where indicated, or DMSO was added as
solvent control. 2 hours before harvest, 5FU was added to a final concentration of
50M where indicated. Luciferase activity was assessed in harvested cell lysates, and
is displayed in arbitrary machine units. Average from triplicates are shown along with
standard deviations.