Supplementary Figures (doc 30K)

Supplementary Figure Legends
Fig. S1. PyMT interacts with Taz and induces cellular transformation of Rat1 cells. A.
PyMT immunoprecipitates with Taz. HEK293 cells were transfected with indicated
constructs and subject to immunoprecipitation with anti-Flag conjugated beads then
blotted using antibodies as indicated. The d2-4PyMT mutant, which does not interact
with Taz, and additional PyMT-signaling complex members, and is inactive in
transforming cells was used here as a negative control. Densitometry of band intensity is
depicted. Band intensities for co-immunoprecipitated PyMT were normalized to Taz
bands. B. PyMT transforms Rat1 cells in focus formation assay. Rat1 cells were
transfected with indicated constructs and cultured until colonies appeared, for 14 days.
Plates were then fixed and stained with Crystal Violet to visualize colonies. The d24PyMT mutant failed to transform Rat1 cells. C. Bright field microscopy of Crystal
Violet stained colonies, as in B. D. Dose-dependent response for colony formation
induced by transfected PyMT in Rat1 cells, using the calcium phosphate method. Bars
represent average number of colonies for each plate in three independent experiments,
error bars represent SEM.
Fig. S2. Characterization of stable cell lines Rat1 Tet-shmTaz-On and Rat1-GFP-Taz. A.
Dose response for reduction of Taz mRNA upon Dox treatment in Rat1-Tet-shTaz-On
stable cell line. Cells were harvested after 48hrs of Dox treatment. Error bars represent
SEM, n=3 B. Colony formation in WT Rat1 cells, not harboring the inducible Taz
shRNA construct, was unaffected by Dox treatment. C. Experiment described in Fig. 1A.
The table depicts number of colonies per plate for each experiment. D. Experiment
described in Fig. 1C. The table depicts number of colonies per plate for each experiment.
E. Ectopic expression of Taz alone in Rat1 cells did not induce cellular transformation in
Rat1 cells.
Fig. S3. Additional controls for the pBS-8xGTIIC-Luc Tead reporter system. A. Dose
dependent response of Taz and Yap co-activation with 8xGTIIC reporter. HEK293 cells
were transfected with indicated constructs and harvested 24hrs post transfection then
subject to luciferase assay. Error bars represent SEM, n=3. B. Co-transfection of Taz with
Lats shows the expected decrease in Taz co-activation, in 8xGTIIC reporter assay, n=3.
C. Dose-dependent response of PyMT inhibition of Taz co-activation, abbreviated in Fig.
2A, n=3. D. Tead-responsive reporter 8xGTIIC signal is diminished by PyMT but not d24PyMT, which does not bind to Taz and other PyMT binding proteins and is
transformation-incompetent. Kinetics of Tead-responsive reporter was examined for
several days as in Fig. 2B.
Fig. S4. GFP-Taz is nuclear excluded in fully grown PyMT-induced colonies. Rat1-GFPTaz stable cells infected with pBP-PyMT-HA retroviral vector, were mixed with naïve
Rat-GFP-Taz cells and cultured on glass coverslips. Once colonies were formed cells
were fixed and stained with anti HA antibody to detect PyMT expression (red). Images of
fields containing colonies, in which GFP-Taz was dramatically cytoplasmic, and fields
between colonies, in which GFP-Taz is detected in the cytoplasm as well as in the
nucleus, were acquired by confocal microscopy, as depicted by cartoon.
Fig. S5. Quantitative assessment of Yap localization using ImageStream technology. A. Yap
was more cytoplasmic in the presence of PyMT. Rat1 cells infected with PyMT or
control vector (pBP) were imaged for immunofluorescent Yap staining in single-cell
resolution. Quantitative assessment of the degree of similarity between localization of
Yap and DRAQ5, a nuclear marker, was estimated using the Bright Detail Similarity R3
feature. B. Yap localization in PyMT-infected cells exhibits less similarity to nuclear
marker than vector-infected cells, indicating that Yap is more cytoplasmic in the presence
of PyMT. Cells counts and statistics for the quantification of nuclear localization are
provided for all cells, and for the subset of cells exhibiting nuclear localization of Yap
(threshold >1.5) C. Examples of cytoplasmic and nuclear localization of Yap fluorescent
images, as acquired by ImageStream.
Fig. S6. PhosTag analysis of Taz phosphorylation by Lats demonstrates hyperphosphorylated Taz. A. HEK293 cells were co-transfected with indicated constructs.
The next day cells were harvested and indicated samples were treated with CIP (Calf
Intestinal Phosphatase). Samples were then subject to SDS-PAGE supplemented with
20uM PhosTag, and Western blot using indicated antibodies. Taz and phosphorylated
Taz species (pTaz) are indicated. LE, long exposure. B-C. Uncropped image for Fig. 6BC includes co-transfection of the constitutively active Src mutant (Y527F), in addition to
the wild type (WT) Src. WT Src functions as the active form, suggesting that Src is
activated under the experimental conditions used.