Supplementary Figure S1. (a) WB analysis of mesenchymal markers in MDA-MB-231,
Hs578T and HMLE (immortalized non-transformed human mammary epithelial cells-control)
cells with anti-Snail1, -Snail2, -ZEB, -E-Cadherin and- GAPDH antibodies. (b) Control for
specificity of anti-MMP14 antibodies used in this study. WB analysis of MDA-MB-231 cells
transfected with siControl (Con) or three different siRNAs against MMP14 (#1-3) with antiMMP14, -GAPDH antibodies. (c) Western blot analysis of MDA-MB-231 cells with antiMMP14 antibodies, pro-form (63 kDa) and furin processed form (54 kDa) are indicated. (d)
Representative images of time-lapse imaging of MDA-MB-231 cells expressing shControl (Con)
or shNEDD9 (N1, N2) upon labeling with CellMask-DeepRed plasma membrane dye. Images
were taken every 5 sec for 30 min total. (e) Quantification of relative fluorescent intensity units
(RFU) in cells as in (d) over the time in the area indicated by white rectangle; graphs are mean
RFU (%) to original 0 min ±S.E.M.; 10 cells per treatment F test was performed for fitted lines p
is not significant (ns). (f-g) Representative images of shControl (Con) and shNEDD9 (N1, N2)
MDA-MB-231 cells expressing mCherry-MMP14 (red) and stained with anti-Rab5 (c) or antiRab7 (d) antibodies, DNA (DAPI); Scale bar is 10µm.
Supplementary Figure S2. (a) WB analysis of shControl (Con) and shNEDD9 (N1, N2) cells
treated with cycloheximide (CXH) (inhibitor of de-novo protein synthesis) for 2 hours with antiNEDD9, -MMP14 and -GAPDH antibodies. (b) Quantification of WBs as in (a), n=3; one-way
ANOVA with Dunnett’s post-hoc analysis *p=0.0106, 0.01 shCon/shN1 or /N2. (c) Confocal 3D
reconstructed immunofluorescent images of shControl (Con) and shNEDD9 (N1, N2) cells
stained with anti-Rab7, Rab11, Lamp1 antibodies (red) and DNA (DAPI); scale bar is 10µm. (d)
Quantification of volumes of Rab7, Rab11 and Lamp1 positive vesicles in cells as in (c). Data
presented as mean vesicular volume/cell as % to shControl (assigned 100%) ±S.E.M; n=3 with
10 cells per treatment; one-way ANOVA with Dunnett’s post-hoc analysis p is non-significant
for shCon/shN1 or/N2 for Rab7, 11 and Lamp1. (e) Representative images of shControl (Con)
and shNEDD9 (N1) cells transfected with RFP-Rab7 (red) during live cell imaging to assess
internalization and trafficking of fluorescently labeled EGF-647 (green) at indicated time points,
demonstrating internalization and delivery of fluorescently labeled EGF to late Rab7 positive
endosomes; scale bar is 10µm. (f-g) Fluorescent pH sensitive dye was added to of shControl
(Con) and shNEDD9 (N1, N2) cells to assess the acidification of the endosomes (red); no
differences in acidification of endosomes between control and shNEDD9 cells were observed;
three independent experiments with 10 cells per treatment; scale bar is 10µm; one-way ANOVA
with Dunnett’s post-hoc analysis p is non-significant for shCon/shN1 or/N2.
Supplementary Figure S3. (a) WB: WCL of shCon and shNEDD9 (N1, N2) MDA-MB-231
cells with specific antibodies indicated on the figure; * marks p130Cas. (b). Quantification of
WBs as in (a); results presented as mean RIU (%) to control ±S.E.M; one-way ANOVA with
Dunnett’s post-hoc analysis p was not significant (ns) for shCon/shN1 or /shN2 for all tested
protein, except NEDD9 *p<0.0001. (c) Representative image of WB analysis with anti-Rab8
antibodies. Left: WB with anti-Rab8, -NEDD9 (2G9), -GAPDH antibodies. (d) Quantification of
relative intensity units (RIU) of WBs as in (c); graphs are mean RIU as % to control (Con,
assigned as 100%) +S.E.M. one-way ANOVA with Dunnett’s post-hoc analysis.
Supplementary Figure S4. (a) Top: pull-down of Active Arf6 from control or NEDD9
overexpressing (+) MDA-MB-231 cells; WB with anti-Arf6 antibodies; bottom: WCL used for
pull down with anti-NEDD9, -Arf6 total and -GAPDH antibodies. (b) Representative image of
WB analysis with anti-Arf1 antibodies. Top: pull-down of Arf1-GTP from shControl (Con) and
shNEDD9 (N1, N2) MDA-MB-231 cells; Bottom: whole cell lysate (WCL) used for pull-down,
WB with anti-Arf1, -NEDD9 (2G9), -GAPDH antibodies. (c) Quantification of relative intensity
units (RIU) of WBs from (b), Arf1-total in WCL and Arf1-GTP in pull-down); n=3; graphs are
mean RIU as % to control (Con, assigned as 100%) +S.E.M. one-way ANOVA with Dunnett’s
post-hoc analysis, ns. (d) WB: WCL of shCon and shNEDD9 (N1, N2) MDA-MB-231 cells with
specific antibodies indicated on the figure, indicating increase in phosphorylation of PIP5K alpha
upon NEDD9 depletion. (e) Transmission electron microscopy of MDA-MB-231 shControl
(Con) and shNEDD9 (N1) cells. Scale bar 0.5 μm. A higher magnifications of perimembrane
area shown in the insets on the right and bottom, marked by white rectangle or dashed lines in
the main panel. (f) Representative images of WB analysis of transient depletion of NEDD9 on
level of Arf6. MDA-MB-231-LN cells were transiently transfected with control (Con) siRNA or
siRNA against NEDD9 (N1, N2). Following 24h incubation cells were subjected to WB analysis
and probed against indicated targets. Quantification of relative intensity units (RIU) of WBs
Arf6-total; n=3; graphs are mean RIU as % to control (Con, assigned as 100%) +S.E.M. one-way
ANOVA with Dunnett’s post-hoc analysis, ns.
Supplementary Figure S5. (a) Representative images of IP with anti-NEDD9 IgG from
HEK293T cells co-transfected with GFP-NEDD9 and Flag-ACAP1, Flag-GIT1, RFP-ASAP2 or
RFP-ASAP3 plasmids; WB with anti-Flag, -RFP and -NEDD9 antibodies.
Suppl. Movie1-3.
NEDD9 depletion does not interfere with bulk internalization of plasma membrane.
Representative sequences of time-lapse imaging of MDA-MB-231 cells expressing shControl
(Suppl.movie1) or shNEDD9 (N1, N2, Suppl. Movie 1 and 2, respectively) upon labeling with
CellMask-DeepRed plasma membrane dye. Images were taken every 5 sec for 30 min total.
Nikon LiveScan SFC swept field inverted microscope equipped with a Perfect focus x-y stage
control and z-axis motor (Nikon), Photometrics QuantEM CCD camera (Photometrics) and using
a 60X Plan Fluor, NA 1.21 objective.
Suppl. Movie4-6.
NEDD9 depletion does not affect internalization MMP14. Representative image sequences of
shCon (Suppl.movie4), shNedd9 (N1, N2, Suppl. Movie 1 and 2, respectively) MDA-MB231cells expressing PA-mCherry-MMP14. Images were taken every 15 sec for 5 min total on
LSM 510 (Zeiss).
Suppl. Movie7-9.
NEDD9 deficiency leads to increase in recycling of MMP14. Representative image sequences
of shCon (Suppl.movie7), shNedd9 (N1, N2, Suppl. Movie 8 and 9, respectively) MDA-MB231cells expressing mCherry-MMP14. Images were taken every 15 sec for 5 min total on LSM
510 (Zeiss).