Legends to Supplementary Figures.
Suppl. Fig. 1. Re-expression of mir-145 affects the growth of the MPM cell lines. A. Left
panels: Proliferation of ctrl and mimic-145 transfected NCI-H2052 (left) and NCI-H28 (right)
cells as assessed by manual counting (hemacytometer). B. Mimic-145 transfection does not
trigger apoptosis. Upper and lower panels. Histograms show the percentage of “dead”
(propidium iodide positive) and “apoptotic” (annexin-V positive) cells as measured by FACS
analysis after transfection of ctrl- and mimic-145. Bars represent mean ± standard deviation of
at least two independent replicates.
Suppl. Fig. 2. Mimic-145 transfection affects wound healing closure of MPM cells. Left
panels: representative micrographs of wound-healing closure assays of MSTO-211H cells
transfected with ctrl- or mimic-145.Scale bar: 100μm. Right panels: histogram shows the
percentage of wound closure at the indicated times. Bars represent mean ± standard deviation
of at least two independent replicates. The p-value refers to matched control vs mimic-145
transfected samples.
Suppl. Fig. 3. Mimic-145 transduction affects ectopically expressed OCT4 and its target
ZEB1. Left panel. Representative western blot analysis of whole cell lysates from control and
mimic-145 transfected cells, either mock-transfected (upper panels) or transfected with an
OCT4 expression vector (retaining the wt 3’UTR region of the gene) (lower panels) and stained
with anti-OCT-4, anti-ZEB1 antibodies or anti-actin as a loading control. Right panel: Histogram
bars represent mean ± standard deviation of at least two independent replicates. The p-value
refers to matched control vs mimic-145 transfected samples.
Suppl. Fig. 4. Mimic-145 transfection affects the number of OCT4 positive cells. A-B. Flow
cytometry analysis of ctrl- and mimic-145-transfected MSTO-211H cells. A. Upper panel.
Representative gatings used for the data acquisition. Please note that a significant shift of the
side scatter in the mimic-145 transfected cells was observed. Lower panel: histogram plots
shows the difference in the median SSC value of ctrl- and mimic-145 transfected cells,
respectively. B. Representative dot plots from the indicated cells stained with an OCT4 specific
antibody (upper panel) and a matched isotype control antibody (lower panel) to evaluate the
aspecific binding. C-D. Mimic-145 transfection opposes the pemetrexed-induced increase of
OCT4 positive cells. C-D. Flow cytometry analysis of ctrl- and mimic-145-transfected MSTO211H cells treated with pemetrexed. A. Upper panel. Representative gating used for the data
acquisition. Please note that no significant shift of the side scatter in the mimic-145 transfected
cells was observed when treated with pemetrexed. Lower panel: histogram plots show the
difference in the median SSC value of ctrl- and mimic-145 transfected cells, respectively. B.
Representative dot plots from the indicated cells stained with a OCT4 specific antibody (upper
panel) and a matched isotype control antibody (lower panel) to evaluate the aspecific binding.
The percentages indicate the number of positive cells.
Suppl. Fig. 5. 5-Azacitidine treatment increases mir-145 levels. A. Representative results of
pyrosequencing assays from MSTO-211H treated with either vehicle- or with 5-Azacitidine B.
Histogram showing the average levels of methylation (overall methylation comprising all the
CpG islands within the mir-145 regulatory regions-as depicted in A) of MSTO-211H and NCIH2052 treated with either vehicle or with 5-Azacitidine. Histogram bars represent mean ±
standard deviation of at least two independent replicates. The p-value refers to matched control
vs mimic-145 transfected samples. C. MSTO-211H cells were treated with vehicle- or 5Azacitidine (10M-96hrs) and the levels of mir-145 analyzed by PCR. Representative
micrograph of a 2% agarose gel is shown. GAPDH was used as a loading control.