Figure Legends
Figure 1. Induction and phosphorylation of p53 and induction of apoptosis in MMCtreated HCT116 cells. (a) HCT116 p53+/+ and p53-/- cells were treated with MMC for 16
and 24 hours. Whole cell lysates were analyzed for p53, p21, MDM2 and
phosphorylation of p53 at Ser15, Ser20 and Ser392 by Western blotting. -actin
expression was used as loading control. (b) Immunostaining of HCT116 p53+/+ and p53-/cells after treatment 16 hours treatment with MMC. Phosphorylation of p53 was detected
by antibodies specific for phosphorylated Ser15 and Ser37. (c). Apoptosis was assessed
by FACS analysis of caspase activity in HCT116 p53+/+ and p53-/- cells treated with
MMC for 16 and 24 hours. HCT116 p53+/+ and p53-/- colon carcinoma cells (provided by
Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore) were grown in IMDM
supplemented with 10% fetal bovine serum, glutamin and gentamycin in 5% CO2 at
37°C. HCT116 cells were grown with or without 10 g/ml mitomycin C to activate
endogenous p53 (Sigma, St. Louis, MI). Western blotting was performed according to
standard procedure using the following primary antibodies: mouse monoclonal anti-p53
(DO-1, Eurogenetics, Tessenderlo, Belgium), mouse monoclonal anti-MDM2 (2A10,
provided by Sonia Lain, University of Dundee), mouse monoclonal anti-p21 (BD
Pharmingen, CA), mouse monoclonal anti--actin (Sigma, St. Louis, MI). For detection
of phosphorylation of p53, rabbit polyclonal anti-p53 Ser15, Ser20 and Ser392 (Cell
Signaling Technology, Danvers, MA) were used. Immunofluorescence staining was
performed as described (Rahman et al., 2005). Caspase activity was measured using
CaspaTag™ Pan caspase kit (Chemicon, Temecula, LA).
1
Figure 2. Pro-QD stained 2D gels showing differential presence of phosphoproteins. (a)
2D gel electrophoresis of HCT116 p53+/+ and p53-/- cell lysates after 16 hours of MMC
treatment. The figure shows a gel section demonstrating phosphorylation of ANXA1
exclusively in the p53+/+ cells after p53 activation. 3D-rendering shows protein spot peak
of phosphorylated ANXA1 in the p53+/+ cells. (b) Multi-channel viewing was applied for
detection of ANXA1 total protein levels by SR staining (red) as well as phosphorylation
status using Pro-QD staining (blue) after MMC-treatment. 2D gel electrophoresis was
carried out as outlined (Rahman-Roblick et al., 2007). Phosphoprotein staining of 2DE
polyacrylamide gels using the Pro-QD phosphoprotein gel stain was performed according
to the manufacturer’s protocol (Molecular probes–Invitrogen, Sweden). The SDS gels
were destained and restained with SR staining solution for total protein staining.
Scanning and image analysis was performed as described previously. PDQuest (Version
8.0.1) was used for semi-automated spot detection and matching. Multichannel viewer
function was applied for gel image overlay. Boolean analysis combining Students t-test
and qualitative analysis defined phosphorylated proteins in the HCT116 cells following
p53 induction. P<0.5 was considered as statistically significant. Statistical analysis was
based on the PDQuest analytical software package. SR-stained protein spots of interest
from the 2D gels were excised using EXQuest spot cutter (Bio-Rad). Manual in-gel
digestion was performed as described (Rahman-Roblick et al., 2007). Peptide mass
fingerprinting was performed in an Ultraflex TOF/TOF instrument (Bruker Daltonics).
Figure 3. Phosphorylation and total protein status of two targets in 2D gel before and
after activation of p53. (a) Upregulation of Nm23-H1 upon p53 activation was detected
by SR staining and phosphorylation was observed by Pro-QD staining of the same gel.
2
(b) Phosphorylation of the DJ-1 protein in p53+/+ cells after MMC treatment. No change
in total expression was detected. White background was applied for better visualization.
Supplementary Figure 4. p53-dependent protein phosphorylation as detected by
immunoprecipitation with the mouse monoclonal anti-pSTT antibody specific for
phosphoserine/threonine/tyrosine (Abcam, Cambridge, UK). Protein-antibody complexes
were captured by protein G Dynabeads (Invitrogen, Oslo, Norway) and analyzed by
Westen blotting using specific antibodies against (a) ANXA1 (mouse monoclonal, BD
Pharmingen, CA; rabbit polyclonal Zymed Laboratories, San Francisco, CA), (b) DJ-1
(rabbit polyclonal, Abcam, Cambridge, UK), or (c) p53 (rabbit polyclonal, Santa Cruz,
CA).
Supplementary Figure 5. ANXA1 and DJ-1 phosphorylation in MMC-treated HCT116
p53+/+ cells upon siRNA knockdown of p53. The cells were transfected with 20nM of
p53 (sip53) or control siRNA (siCtrl) (Qiagen, Cambridge, MA) 24 hours prior to MMC
treatment. (a) Phosphorylation of ANXA1 and (b) DJ-1 was assessed by
immunoprecipitation and Western blotting as described in Fig. 4. (c) Western blot
analysis confirmed absence of upregulation of p53 and MDM2 in the sip53-treated cells.
Supplementary Figure 6. Phosphorylation of DJ-1 in MMC-treated HCT116 p53+/+
cells upon inhibition of transcription by actinomycin D. Cells were treated for 2 hours
with 100nM actinomycin D (actD; Sigma, St. Louis, MI) prior to MMC treatment. (a)
Expression of the p53 targets p21 and MDM2 was assessed by Western blotting. -actin
3
was used as loading control. (b) Phosphorylation of DJ-1 and (c) p53 was detected as
described above.
Supplementary Figure 7. ANXA1 and DJ-1 phosphorylation in MMC-treated HCT116
p53+/+ cells upon siRNA knockdown of JNK1. The cells were transfected with 40nM of
JNK1 (siJNK1) or control siRNA (siCtrl) (Qiagen, Cambridge, MA) for 48 hours prior to
MMC treatment. (a) Phosphorylation of ANXA1 and DJ-1 was assessed by
immunoprecipitation and Western blotting as described in Fig. 4. (b) Real time PCR was
performed according to the manufacturer’s instructions to confirm downregulation of
JNK1 in the siJNK1-treated cells (Applied Biosystems, Foster, CA).
4