1. Volume 26, Issue 6, Pages 658-665 (September 2013)
YAP and TAZ, Hippo Signaling Targets, Act as a Rheostat for Nuclear SHP2 Function 
Ryouhei Tsutsumi, Mohammad Masoudi, Atsushi Takahashi, Yumiko Fujii, Takeru Hayashi, Ippei Kikuchi, Yumeko Satou, Masanori Taira, Masanori Hatakeyama 
Developmental Cell 
Volume 26, Issue 6, Pages (September 2013)
DOI: /j.devcel
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2. Developmental Cell 2013 26, 658-665DOI: (10.1016/j.devcel.2013.08.013)
Copyright © 2013 Elsevier Inc. Terms and Conditions

3. Figure 1 Cell-Density-Dependent Subcellular Distribution of SHP2
(A) AGS cells cultured at low density were treated with indicated reagents and were immunostained with an anti-SHP2 antibody. The graph shows the ratio of SHP2 intensity in the nucleus to that in the cytoplasm. Error bars indicate mean ± SD; n = 5. ∗∗p < FBS, fetal bovine serum.
(B) AGS cells cultured at low density in the presence or absence of 0.5 μg/ml C3 were stained with an anti-SHP2 antibody and DAPI. Scale bar represents 50 μm.
(C) AGS cells cultured at low density were transiently transfected with the indicated Rho vector and were stained with an anti-SHP2 antibody. Scale bar represents 50 μm.
(D) AGS cells cultured at high density in the presence or absence of 20 nM staurosporine were stained with an anti-SHP2 antibody and DAPI. Scale bar represents 50 μm.
(E) Schematic views of SHP2 mutants used in this study. Asterisks indicate the evolutionally conserved proline residues that are mutated in the SHP2-PA mutant.
(F–H) AGS cells cultured at low density were transfected with the Myc- or Flag-tagged SHP2 vector and were stained with an anti-Myc antibody (F and G) or an anti-Flag antibody (H). Nuclei were visualized by DAPI. The box plots show the ratio of SHP2 intensity in the nucleus to that in the cytoplasm. Boxes include 25th−75th percentiles with bars indicating medians; whiskers indicate range of data other than outliers represented by dots. n = 60. ∗∗∗p < Scale bars represent 50 μm.
See also Figure S1.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

4. Figure 2
YAP and TAZ Regulate Subcellular Distribution of SHP2 in a Hippo Signal-Dependent Manner
(A) AGS cells cultured in a low-density or high-density condition were stained with anti-SHP2 antibody, anti-YAP/TAZ antibody, phalloidin, and DAPI. The box plots show the ratio of SHP2 intensity in the nucleus to that in the cytoplasm. Boxes include 25th−75th percentiles with bars indicating medians; whiskers indicate range of data other than outliers represented by dots. n = 30. ∗∗∗p < Scale bar represents 50 μm.
(B and C) HEK293T cells were transfected with expression vectors of either HA-YAP or HA-TAZ, together with that of Flag-SHP2. Anti-Flag (B) or anti-HA (C) immunoprecipitates (IP) from cell lysates and total cell lysates (TCL) were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Relative intensity value shown below each of the SHP2 bands was determined as the ratio of intensity of the coprecipitated Flag-SHP2 band to that of the immunoprecipitated HA-TAZ using a luminoimage analyzer.
(D) Purified recombinant glutathione S-transferase (GST)-TAZ or GST was mixed with recombinant SHP2 and was subjected to an in vitro binding assay.
(E–I) AGS cells cultured in a low-density (E and F) or high-density (H and I) condition were transfected with YAP/TAZ-specific shRNA vectors (E and I), HA-tagged LATS2 vector (F and G), or HA-tagged wild-type TAZ or TAZ-S89A vector (H). Cells were fixed and stained with an anti-SHP2, anti-YAP/TAZ, or anti-HA antibody. Arrows indicate cells in which HA-LATS2 (F) or HA-TAZ (H) was ectopically expressed. Nuclei were visualized by DAPI. An RNAi-resistant HA-TAZ vector was cotransfected with the shRNA vector for a rescue experiment (E). Lysates of cells expressing HA-LATS2 were subjected to immunoblotting with respective antibodies. The numbers indicate relative intensities of the YAP or TAZ band (G). The box plots show the ratio of SHP2 intensities in the nucleus to that in the cytoplasm. Boxes include 25th−75th percentiles with bars indicating medians; whiskers indicate range of data other than outliers represented by dots. n = 30. ∗∗∗p < Scale bars represent 50 μm.
See also Figure S2.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions

5. Figure 3
YAP and TAZ Regulate Nuclear Function of SHP2 in a Hippo Signal-Dependent Manner
(A) AGS cells cultured in a low-density condition were transfected with the YAP/TAZ shRNA expression vectors or control vector as indicated. Endogenous parafibromin (PF) was immunoprecipitated from cell lysates. Immunoprecipitates (IP) and total cell lysates (TCL) were subjected to immunoblotting with indicated antibodies. The numbers above the αPF lanes indicate relative intensities of tyrosine-phosphorylated parafibromin (pY-PF) that were normalized to the levels of immunoprecipitated parafibromin.
(B–H) AGS cells cultured in low- and high-density conditions (B, C, and F) or in a low-density condition (D, E, G, and H) were transfected with indicated vectors and reporter plasmids. Relative luciferase activities are shown in graphs with the value in control cells calculated as one. Total cell lysates were analyzed by immunoblotting with indicated antibodies. Error bars indicate mean ± SD; n = 3. ∗∗∗p < , ∗∗p < In (D), protein bands shown were visualized on identical films with the same exposure times for left and right images.
(I) RT-qPCR analysis of NKD1 and AXIN2 mRNA expression in GES-1 cells transiently transfected with a TAZ-S89A vector. Error bars indicate mean ± SD; n = 3. ∗∗∗p <
(J) AGS cells cultured in a low-density condition were transfected with indicated vectors and reporter plasmids. Relative luciferase activities are shown in graphs with the value in control cells calculated as one. Total cell lysates were analyzed by immunoblotting with indicated antibodies. Error bars indicate mean ± SD; n = 3. ∗∗∗p <
(K) Ptpn11fl/KO MEFs treated with 4-hydroxytamoxifen (4-OHT) were transfected with a TAZ-S89A vector and reporter plasmids. Relative luciferase activities are shown in graphs with the value in control cells calculated as one. Error bars indicate mean ± SD; n = 3. ∗∗∗p < , ∗∗p < 0.001.
(L) Immunohistochemistry of YAP/TAZ and SHP2 expression in the colon of a normal mouse. Squares indicate areas presented at higher magnification. Scale bars represent 10 μm.
Developmental Cell  , DOI: ( /j.devcel )
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6. Figure 4
SHP2 Potentiates Transcriptional Coactivator Function of YAP/TAZ
(A–C) AGS cells cultured in a low-density (A–C) or high-density (A) condition were transfected with indicated vectors together with reporter plasmids. Total cell lysates were subjected to immunoblotting with indicated antibodies. Relative luciferase activities are shown in graphs with the value in control cells calculated as one. Error bars indicate mean ± SD; n = 3. ∗∗∗p <
(D) Ptpn11fl/KO MEFs treated with 4-OHT were transfected with TAZ-S89A and reporter plasmids. Total cell lysates were analyzed by immunoblotting with indicated antibodies. Relative luciferase activities are shown in graphs with the value in control cells calculated as one. Error bars indicate mean ± SD; n = 3. ∗∗∗p <
(E) RT-qPCR analysis of Ctgf and Cyr61 mRNA expression in Ptpn11fl/KO MEFs transiently transfected with a TAZ-S89A vector. Error bars indicate mean ± SD; n = 3. ∗∗∗p < 
(F) NIH 3T3 cells transfected with the TAZ-S89A vector together with the SHP2 shRNA vector were subjected to a soft-agar assay. Relative colony numbers are shown in graph. Error bars indicate mean ± SD; n = 3. ∗∗p <
See also Figure S3.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2013 Elsevier Inc. Terms and Conditions