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Malaterre et al., 2015
Supplementary Information
Submission to Oncogene
Legends for Supplementary Figures
Supplementary Figure 1. Representation of the MybERT2 transgene DNA sequence
and Tamoxifen-activation in reporter assays. (a) Sequence of the MybER transgene is
shown highlighting the ATG translation start site and the position of the Sma1 restriction
endonuclease site that serves as the fusion junction allowing the protein to fold when not
exposed to Tamoxifen. (b) Chloramphenicol acetyl-transferase assays (CAT) were
conducted in triplicate to show activation of a Myb-responsive reporter in response to
the addition of 4-hydroxy-tamoxifen (4OHT) shows acetylation of the substrate as
detailed elsewhere (5). (c) The hinge region between the fusion partner and the ER
domain is critical to optimal activation accordingly several fusions were explored to
generate a tightly controlled inducible MybER construct. We found that creating a
junction at the SmaI site was superior to a nearby Xho1 site in CAT reporter assays
compared to vector alone controls, with and without 4OHT.
Supplementary Figure 2. Myb-activation is required for the initiation and progression of
early stage adenomas. (a) Colonic adenomas were isolated, dissociated into single cells
and embedded in Matrigel. Induction of MybER was achieved by supplementing cultures
with 4OHT during the entire duration of the experiment (initiation and progression) or
during the first 48hr (initiation phase) or after 48hr (after initiation phase). (b) MybER is
required to be active at least during the first 48hr of initiation of the culture to significantly
increase the number of organoids. (c) MybER activation after the first 48hr of initiation of
organoid growth did not increase the number of adenomas organoid formed. (d)
Sustained MybER activation by 4OHT was required to achieve optimal growth while
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Malaterre et al., 2015
Supplementary Information
Submission to Oncogene
4OHT withdrawal ameliorated the growth of adenoma organoids. Data presented for
individual samples with means +/- SEM; *P<0.05.
Supplementary Figure 3. Short-term activation of MybER does not affect colonic crypt
homeostasis. MybER transgenic mice were fed Tamoxifen to activate the transcription
factor fusion protein where after mice were culled and colons assessed. (a) Micrographs
of colons from WT and MybER line#3 following Tamoxifen being provided in chow for 0,
3 or 6 days. (b) Number of PCNA+ve nuclei per crypt is shown. (c) Myb target gene
CyclinE1 (Ccne1) was evaluated in three transgenic lines where the transgene was
transmitted to show elevated expression in lines 2 and 4. Data presented for individual
samples with means +/- SEM; *P<0.05, **P<0.01.
Supplementary Figure 4. Pten is induced in MybER-activated colonic crypts and this is
reversed in mice on a p27 heterozygous background. (a) Pten was detected by IHC on
colonic crypt section. Nuclear Pten was prominent at the base of the crypt. (b) Positive
Pten cells were quantified showing that Pten was significantly elevated in MybER mice
but not in p27+/-:MybER mice.
Supplementary Figure 5. Heterozygote loss of the cell cycle inhibitor p27 gene
increases the number of cells expressing CyclinE1 in colon crypts of p27+/-:MybER mice
compared to MybER mice. (a) p27 expression was detected in colonic crypts using IHC.
P27 is mainly expressed in IPC zone (black arrow) compare to ISC zone at the very
bottom of the crypts (white arrow). Note that some cells strongly express p27 in MybER
colonic crypts (red arrow). p27 expression was reduced in crypts of p27 mice compared
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Malaterre et al., 2015
Supplementary Information
Submission to Oncogene
to WT. CyclinE1 was detected by IHC in colonic crypts. (b) p27 heterozygous loss
resulted in the increase number of CyclinE1 positive ISC/IPC cells. Data presented are
mean +/- SEM of at least 3 different samples. (c) p27 was detected by IHC and
quantified. As expected p27 was reduced in both p27 heterozygous and p27+/-:MybER
mice. The number of positive p27+ve cells was not different between wt and MybER
mice. One-way ANOVA;*P<0.05.; **P<0.01;Scale bar is 50M.
Supplementary Figure 6. Intestinal homeostasis is not changed by MybER-activation
alone but its effect is revealed upon heterozygous loss of the cell cycle inhibitor p27
gene. MybER mice were fed Tamoxifen chow for 2 weeks and cell proliferation was
assessed using IHC for the proliferation marker Proliferating Cell Nuclear Antigen
(PCNA). PCNA+ve cells were scored according to their location within the crypt with “0”
allocated to the crypt base where putative stem cells are located. (a) We found no
significant changes in the number of PCNA+ve cells in the colon of Tamoxifen treated
MybER mice compared to Tamoxifen treated WT mice. (b) When MybER was activated
on a p27+/- background we found a significant difference in p27+/-:MybER compared to
WT mice while no significant difference could be seen in p27+/- compared to WT mice.
(c) When MybER was activated in Apcmin/+ mice it did not have any additive effect on cell
proliferation compared to Apcmin/+ alone. (d) Assessment of the total number of PCNA+
cells per colonic crypt showed a significant increase in cell proliferation in Apcmin/+ mice
compared to WT mice and in p27+/-:MybER compared to either to p27+/- mice or WT
mice. Data presented are mean +/- SEM of at least 3 different samples. *P<0.05,
**P<0.01 and ***P<0.001. (e) Representative fields of view of colon sections stained for
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Malaterre et al., 2015
Supplementary Information
Submission to Oncogene
PCNA from Tamoxifen-treated WT, MybER, Apcmin/+, Apcmin/+:MybER, p27+/- and p27+/:MybER mouse groups.
Supplementary Figure 7. MybER activation increases AOM-induced ACF and CRC.
To investigate a direct role of c-Myb in early colon tumorigenesis we employed the procarcinogen AOM that has a strong tropism for the colon inducing the formation of
adenomas. Mice were treated with Tamoxifen for two weeks prior to AOM injection (one
weekly IP injection over six weeks). (a) Mouse cohorts on three different genetic
backgrounds (WT, p27+/- and Apcmin/+) were harvested when the first mouse (4, 10 or 18
wks) of each group showed signs of disease and abnormal crypt foci (ACF) were
quantified numerically by methylene blue staining, (b) according to size (crypts per
focus) and (c) by the percentage of ACFs that belong to defined size classes via
counting the number of abnormal dysplastic crypts per ACF on H&E stained paraffin
sections. (d) The number and size (e) of tumors were also assessed. Although mice in
an Apcmin/+ background became sick earlier with a higher burden of ACFs and CRCs the
presence of the MybER transgene in some instances was associated with more or
larger ACF but this did not reach statistical significance. Data presented for individual
samples with means +/- SEM.
Supplementary Figure 8. MybER activation in adenomas influences markers of
angiogenesis and proliferation. (a) Hif1 expression is increased in MybER mice as
assessed by IHC. (b) Examination of adenomas for neo-angiogenesis at the interface
with the muscularis does not reveal elevated CD31+ve endothelium (c,d), or increased
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Malaterre et al., 2015
Supplementary Information
Submission to Oncogene
PCNA staining (e) determined by area counted as pixels. Data presented for individual
samples with means +/- SEM are shown.
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