Supplementary Figure legends
Supplementary Fig. 1. Semi-quantitative RT-PCR for miR-29b and BCL9L
HEK293 cells were transiently transfected with pEGFP-N3 (Control) or pEGFP-N3-miR-29b plasmid.
After 24h, the cells were treated with 25mM LiCl for 18h and analyzed for expression of miR-29b,
RNU6 (internal control), BCL9L and GAPDH (internal control) by SQ-RTPCR as described in
Materials and methods. The same results were observed in another independent experiment.
Supplementary Fig. 2. Effect of ectopic expression of miR-29b on GSK3B and TP53 in SW480
cells
(A) Total lysates of SW480 cells transfected with control oligo or 80nM miR-29b mimic were prepared
48h post transfection and subjected to SDS/PAGE and immunoblotting for detection of GSK3B,
Phospho-GSK3B (pSer9) and ACTB. (B) Total lysates of SW480 cells transfected with control oligo or
miR-29b mimic (20nM, 40nM or 80nM) were prepared 24h post transfection and subjected to
SDS/PAGE and immunoblotting for detection of TP53 and ACTB.
Supplementary Fig. 3. Effect of ectopic expression of miR-29b on BCL9L expression and
CTNNB1 localization in SW480 cells
(A) Whole lysates of SW480 cells transfected with control oligo or miR-29b mimic (20nM, 40nM or
80nM) were prepared 48h post transfection and subjected to SDS/PAGE and immunoblotting for
detection of BCL9L and ACTB. (B) SW480 cells transfected with control oligo or miR-29b mimic were
fixed 24h after transfection, permeabilized and stained with anti-CTNNB1 primary antibody followed
by rhodamine-conjugated secondary antibody. DAPI was used for nuclear staining. Images were
taken at 20x magnification using Nikon Eclipse TS-100 microscope. Red fluorescence was detected
using G-2A fluorescence filter set and DAPI using the UV-2A filter.
Supplementary Fig. 4. Effect of ectopic expression of miR-29b on adhesion of SW480 cells to
plastic
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SW480 cells transfected with control oligo or miR-29b mimic were reseeded on uncoated
bacteriological plates and live and dead cells were counted at 48h in the adherent and suspension
fractions after trypan blue staining (n=4, mean ± S.E; **P<0.01 vs. control).
Supplementary Fig. 5. Effect of ectopic expression of miR-29b in SW480 cells on TCM-induced
in vitro tube formation of ECV304 cells
ECV304 cells were suspended in the TCM collected from SW480 cells transfected with control oligo
or miR-29b mimic and seeded on matrigel in 96 well plates and images were taken under microscope
at 4x magnification after 18h incubation. Total number of tubes and branching points and total tube
length in each sample were estimated using WimTube (WimasisGmbH) online analysis tool (n=3,
mean ± S.E; **P<0.01/*P<0.05 vs. control).
Supplementary Fig. 6. Effect of ectopic expression of miR-29b in SW480 cells on the TCMinduced proliferation and migration of EA.hy926 cells
(A) EA.hy926 cells were suspended in the TCM collected from SW480 cells transfected with control
oligo or miR-29b mimic, DMEM or cDMEM and seeded in wells with or without GFR-matrigel in 96
well plates. Cell viability was assayed by resazurin reduction after 18h incubation (n=3, mean ± S.E).
(B) EA.hy926 cells were grown as a monolayer and a scratch was introduced in the monolayer. The
cells were then incubated with SW480 TCM from control oligo or miR-29b transfected cells for 18h.
The cell migration was estimated as scratch area closure using WimScratch (Wimasis GmbH) online
analysis tool and expressed as percentage of control (n=3, mean ± S.E).
Supplementary Tables
Supplementary Table 1
Primers for amplification of pri-mir-29a/b/c and BCL9L 3’UTR
Supplementary Table 2
Primers for semi-quantitative/quantitative PCR
Supplementary Table 3
Primers for reverse transcription and semi-quantitative/quantitative PCR of miR-29b and RNU6
Supplementary Table 4
Predicted and validated targets for miR-29 family involved in the regulation of Wnt signaling
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