Supplemental figure legends:
Figure S1: VEGF up-regulation in ALK oncogene-expressing conditional
transgenic mice. (A) Macroscopic analysis of onco-ALK-positive (ALK+) transgenic
mice shows a robust angiogenesis when compared to onco-ALK-negative (ALK-) and
normal littermate transgenic mice (tTA). (B) RT-qPCR assays were performed to
determine VEGF mRNA levels in lymph nodes of transgenic mice, under both the
ALK+ and ALK- conditions. VEGF mRNA amounts were first normalized to the
amounts detected in lymph nodes from tTA controls. (C) ELISA assays were
performed on indicated mice sera to quantify secreted VEGF. Each experiment was
performed in triplicate and repeated three times. Bars represent standard deviation
and asterisks depict statistically significant differences (*: p<0.05; Student's t-test).
The number of mice in each group is denoted by the n value.
Figure S2: MiR-16 overexpression following transfection into TPM3-ALK MEF
cells.
MiRNA-16 levels were quantified by RT-qPCR, with normalization to
SnoRNA202, in MEF TPM3-ALK cells transfected with pre-miR-16 (miR-16) or a
scrambled pre-miRNA control (Sc). Bars represent standard deviation and asterisks
depict statistically significant differences compared to the scrambled control (**:
p<0.01; Student's t-test). Each experiment was performed in triplicate and repeated
three times.
Figure S3: Expression of miR-16 is not epigenetically regulated in human ALKpositive ALCL. MSP analysis of the human miR-16-1 (hsa-miR-16-1/DEUL2 host
gene) and miR-16-2 (has-miR-16-2/SMC4 host gene) in 14 ALK+ ALCL biopsies
(ALCL-10, -21, -22, -25, -27, -29, -32, -33, -35, -37 to -40 and -53). MC; positive
methylated control; UC: non methylated control; H2O: water blanks.
Figure
S4:
Expression
of
HIF-1
in
human
ALK-positive
ALCL.
Immunohistochemistry (immunoperoxidase staining, original magnification x400/0.85)
was performed to determine HIF-1 expression in 4 human ALK+ ALCL (ALCL-22, 27, -33 and -39).
Supplemental table legends:
Table S1: Northern-blotting quantification of miR-15 and miR-16 expression in
lymph nodes from onco-ALK conditional transgenic mice.
Table shows
expression levels of ALK oncogenes (TPM3-NPM and NPM-ALK) in the absence
(ALK+, n=2 for each oncogene) or presence (ALK-, n=2 for each oncogene) of
doxycycline. Values were normalized to the one obtained using lymph nodes from
two littermate mouse controls. Non-parametrical Mann-Whitney test and unpaired t
test were applied to analyze the differences between groups.
Table S2: Primers used for methylation-specific polymerase chain reaction