NF-kB-mmc1

Cancer Cell, Volume 23

Supplemental Information

Mutant p53 Prolongs NF-?B Activation

and Promotes Chronic Inflammation

and Inflammation-Associated Colorectal Cancer

Tomer Cooks, Ioannis S. Pateras, Ohad Tarcic, Hilla Solomon, Aaron J. Schetter, Sylvia Wilder, Guillermina Lozano, Eli Pikarsky, Tim Forshew, Nitzan Rozenfeld, Noam Harpaz, Steven Itzkowitz, Curtis C. Harris, Varda Rotter, Vassilis G. Gorgoulis, and Moshe Oren

Supplemental Data

? Figure S1, related to Figure 1

? Figure S2, related to Figure 2

? Figure S3, related to Figure 3

? Figure S4, related to Figure 5

? Figure S5, related to Figure 6

? Figure S6, related to Figure 7

? Figure S7, related to Figure 8

Supplemental Experimental Procedures

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Supplementary data

Figure S1, related to Figure 1. NF-κB activation by TNF-α is extended by mutp53

A. PANC-1 cells were transfected with the indicated combinations of siRNA oligonucleotides directed against p53, p65 or scrambled control (Con). 48 hours later, cells were either treated with 2

0.5 ng/ml TNF-α for an additional 24 hours (+) or left untreated (-). Cell extracts were subjected to Western blot analysis with the indicated antibodies.

B. PANC-1 cells were stably transfected with p53 shRNA (shp53) or LacZ shRNA as control (shLacZ), or transiently transfected with either a pool of four p53 siRNA oligonucleotides (sip53) and their corresponding control siRNA (siCon), or with a single p53 siRNA (sip53II) and its corresponding control siRNA (siConII). Cultures were exposed to 0.5 ng/ml TNF-α for 24 hours, at which time RNA was extracted and subjected to qRT-PCR analysis with primers specific for p53 (white bars) or IL-8 (black bars). Values were first normalized for GAPDH mRNA in the same sample, and then each p53 knocked-down sample was compared to its corresponding control sample, defined as 1.0.

C. PANC-1 cells were transiently transfected with p53 siRNA or control siRNA, and exposed to 0.5 ng/ml TNF-α for 24 hours. RNA was extracted and subjected to qRT-PCR analysis with primers specific for the indicated genes. Values were calculated as in (B).

D. PANC-1 cells stably transfected with p53 shRNA (shp53) or control shRNA (shLacZ) were transiently transfected with a luciferase reporter gene driven by 3 tandem NF-?B responsive elements. Cultures were exposed to 0.5 ng/ml TNF-α for 24 hours, and then cells were harvested and luciferase activity was determined. Values were normalized to Renilla luciferase readings in the same samples.

E. PANC-1 cells were transfected with siRNA oligonucleotides (SMARTpool) directed against p53 (sip53) or with random control siRNA (siCon), and 48 hr later were treated either with TNF-α (TNF, 0.5 ng/ml) or PBS (No). Following 24 hr of TNF-α treatment, RNA was extracted and subjected to expression microarray analysis. The heatmap depicts the expression patterns of the genes whose abundance was significantly upregulated or downregulated following addition of TNF-α. For each row (gene) the log-expression values were centered (mean 0) and normalized 3

(STD = 1). Red and blue colors indicate increased and decreased expression, respectively, relative to the mean value of a gene.

In the entire figure, bars represent +/- SD.

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Figure S2, related to Figure 2. Mice expressing mutp53 are excessively susceptible to DSS

A. Mouse embryonic fibroblasts (MEFs) were prepared from mice of different p53 genotypes (WT = +/+, null = -/-, m/m = homozygous mutant, +/m = heterozygous), passaged minimally in culture, and then exposed to the indicated concentrations of TNF-α. RNA extracted from cells harvested at the indicated time points was subjected to qRT-PCR analysis with primers specific for KC mRNA. Values were normalized to GAPDH

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