Fig. S1. p53 DNA contact mutants exhibit a stronger induction of the CGS

compared to the p53 conformational mutants

Six human tumor-derived cell lines that endogenously express different p53 mutants

and vary in their Ras status were stably infected with shRNA against p53 (shp53) or

with a control shRNA (shCon). (A) Table summarizing p53 and Ras status in the

cells lines. (B) mRNA levels of p53 were measured by Quantitative Real Time PCR

(QRT-PCR). (C) mRNA levels of representative CGS genes CXCL1, IL-1β and IL-8

were measured by QRT-PCR. shCon/shp53 fold changes were calculated and plotted

on graphs. Black bars represent lines expressing a p53 DNA contact mutants and gray

bars represent cell lines expressing p53 conformational mutants.

Fig. S2. p53 Zn+2 region conformational mutants expressing cells harbor elevated

levels of activated H-Ras

Ras activity pull down assay was carried out in the indicated WI-38 cell lines using

beads that are fused to Ras binding domain (RBD) via glutathione S-transferase

(GST) (GST-RBD). (A) Western blot analysis depicts the protein levels of active Ras,

total Ras, p53 and GAPDH as a loading control. (B) Quantification of the H-Ras-GTP

bands, normalized to β-tubulin, analyzed by ImageJ software.

Fig. S3. The wild-type p53 transcriptional target, p21WAF1, is not affected by the

expression of the various p53 mutants

(A) mRNA levels of the wild-type p53 target gene, p21WAF1, were measured by

Quantitative Real Time PCR (QRT-PCR) in the established WI-38 cell lines. (B)

H1299 p53-null cell line was transiently transfected with the following expressing

vectors: control empty vector (Con), wild-type p53 (p53 WT), p53R175H, p53G245S,

p53R248Q. Following 48 hours of transfection cells were collected and mRNA levels of

p21WAF1 were measured by QRT-PCR.

Fig. S4. The various mutant p53 forms attenuate wild-type p53 activity by a

dominant negative mechanism following DNA-damage

WI-38 cells were treated with cisplatinum (1µg/ml) or with DMSO (as a control) for

72 hours. p21WAF1 and BTG2 mRNA levels were measured by Quantitative Real Time

PCR in the indicated WI-38 cell lines.

Fig. S5. NFκB mediates the elevated induction of the CGS expression exerted by

the p53 DNA contact mutants

The indicated WI-38 cell lines were transfected with either si-RNA against p65 or

with a control si-RNA (si-Con). Cell were collected 72 hours post transfections. (A)

p65 protein levels were measured by western blot analysis. (B) CXCL1, IL-1β and

MMP3 mRNA levels were measured by Quantitative Real Time PCR (QRT-PCR).

The number above each bar represents the fold repression in each individual cell line

caused by p65 knock-down. (C) SW-620, SW-480 and SKBR-3 cell lines were

knocked-down for the endogenously expressed mutant p53. Western blot analysis

depicting the protein levels of p53 and the GAPDH housekeeping control protein in

the established cell lines. (D) The established SW-620 cells were transfected with

either si-RNA against p65 or with a control si-RNA (si-Con). p65 mRNA levels were

measured by QRT-PCR.

Fig. S6. The CGS levels are not elevated by the p53 conformational mutants

under basal conditions or TNF-α treatment

NCI-H322 and Hs-578-T cell lines, which endogenously express p53 conformational

mutants, were stably introduced with shRNA against p53, and then treated with

20ng/ml TNF-α for 24 hours. mRNA levels of p53, CXCL1, CXCL2 and IL-8 were

measured by Quantitative Real Time PCR.

Tables

Table S1: Primers for QRT-PCR:

Genes and promoters Sense Anti-sense

GAPDH ACCCACTCCTCCACCTTTGA CTGTTGCTGTAGCCAAATTCGT

CXCL1 AGTCATAGCCACACTCAAGAATGG GATGCAGGATTGAGGCAAGC

IL-1β GCCTGAAGCCCTTGCTGTAGT GCGGCATCCAGCTACGAAT

MMP3 ACAAAGGATACAACAGGGACCAA CAATTTCATGAGCAGCAACGA

BTG2 AGGCACTCACAGAGCACTACAAAC GCCCTTGGACGGCTTTTC

p21WAF GGCAGACCAGCATGACAGATT GCGGATTAGGGCTTCCTCT

H-Ras GCTGCATGAGCTGCAAGTGT CATCCGGCACCTCCATGT

p65 ACGAACTGTTCCCCCTCATCT TCCTTTCCTACAAGCTCGTGG

CXCL2 CGCCCAAACCGAAGTCATAG AGACAAGCTTTCTGCCCATTCT

IL-8 GGCAGCCTTCCTGATTTCTG CTTGGCAAAACTGCACCTTCA

Table S2: Primers for site directed mutagenesis:

Mutation Sense Anti-sense

p53R175H ATGACGGAGGTTGTGAGGCACTGCCCCCACCATGA

GCGCT

AGCGCTCATGGTGGGGGCAGTGCCTCACAACCTCC

GTCAT

p53H179R AGGCGCTGCCCCCACCGTGAGCGCTGCTCAGATAG

CGATG

CATCGCTATCTGAGCAGCGCTCACGGTGGGGGCAG

CGCCT

p53G245S TTCCTGCATGGGCAGCATGAACCGGAGGCCCATCC

TCACC

GGTGAGGATGGGCCTCCGGTTCATGCTGCCCATGC

AGGAA

p53R248Q GGCGGCATGAACCAGAGGCCCATCCTCACCATCAT

CACAC

GTGTGATGATGGTGAGGATGGGCCTCTGGTTCATGC

CGCC

p53R273H GGAACAGCTTTGAGGTGCATGTTTGTGCCTGTCCTG

GGAG

CTCCCAGGACAGGCACAAACATGCACCTCAAAGCT

GTTCC