Supplementary materials
Tissue specimen, RNA extraction and PCR analysis
Exclusion criteria were patients treated with adjuvant therapy and with tumor microsatellite
instability (MSI). Histopathological and clinical findings were scored according to the tumornode-metastasis (TNM) staging system of the International Union Against Cancer (Sobin L,
Wittekind C (2002) International Union Against Cancer (IUCC): TNM classification of
malignant tumors. 6th ed. New York: Wiley). Before RNA and DNA extraction, a frozen
section was cut and stained with hematoxylin and eosin to evaluate the percentage of
neoplastic cells. Except for 3 patients, malignant cells accounted for at least 50 % of the
tumor. The study was approved by the National Institute of Cancer (INCa) following the
recommendations of the National Agency of Agreement and Evaluation for Health (ANAES).
Tumor samples have been collected in agreement with the 2004 French Bioethic law.
For RNA extraction, 40 to 80 (5 to 10 µm thick) frozen sections from tumoral and adjacent
normal samples were immediately homogenized in the lysis buffer of the RNeasy extraction
kit and total RNA were extracted according to the manufacturer’s instruction (Qiagen). The
quality of total RNA was assessed on the Agilent 2100® bioanalyser using the RNA
NanoLab® chip and RNA 6000 Nano Assay® kit (Agilent Technologies). 1 to 2 µg of total
RNA were reverse transcribed using the High-Capacity cDNA Archive Kit (Applied
Biosystems). cDNA of Pol and Rev3 transcripts have been amplified in triplicate in tumoral
and normal samples using gene expression assays (ABI cat# Hs00172491_m1 and
Hs00161301_m1 respectively), TaqMan Universal PCR Master and the 7900HT fast real time
PCR apparatus. Four control cDNA (18S, GAPDH, HPRT, YWHAZ) were simultaneously
amplified (gene expression assay : ABI cat# Hs99999901_s1, Hs99999905_m1,
Hs99999909_m1, Hs00237047_m1). All Q-PCR measurements were triplicated and the 3 CT
values do not vary from the mean for more than 1.2%. For each pair of samples, the two most
stable control genes were selected by qBases software (http://medgen.ugent.be/qbase/) to
normalized the expression of Poland Rev3 then ratios T (tumoral)/ N(normal) between
normalized values were calculated.
Statistical analysis
In order to test whether a 2-fold change in gene expression was relevant, we realized a
bilateral exact binomial test by choosing arbitrary as the null hypothesis that “tumoral tissue
underwent a mean 2-fold change in gene expression when compared to the normal one”.
When the p value for this test was higher than 0.05, it reflected a situation approaching the
null hypothesis i.e. a mis-expression ratio close to the significant factor 2. As the statistical
analysis revealed a p=0.5610 for Rev3/pol it indicated that Rev3/pol  was on average 2fold under-expressed in tumors compared to normal tissues. Moreover, it has to be noted that
while 21.6 % (16/74) tumours displayed a more than 4-fold decrease of Rev3 expression in
tumors when compared to normal adjacent tissues, such unbalance was never observed in the
case of Pol.
We observed that the T/N ratio was significantly lower than 1 whatever the stage of the
adeno-carcinomas (not shown). We next investigated whether the level of variation was
different between stages I - II and stages III – IV. In each group 37 cases were considered :
the mean values were 0.545 (min: 0.05; max: 1.94 ) for the stage I-II and 0.537 (min: 0.05;
max: 1.65 ) for stage III-IV. A rank sum test was applied to compare the T/N values between
stages I - II versus stages III – IV and no significant difference was observed (p value= 0.91).
Legend to figure S1
Protein expression of Rev3 was detected by indirect immunofluorescence (red) in growing primary
human mesenchymal stem cells permeabilized with 0.5% triton. DNA was stained with Hoechst
33258.