1. Volume 63, Issue 1, Pages 156-166 (July 2016)
TGF-β Targets the Hippo Pathway Scaffold RASSF1A to Facilitate YAP/SMAD2 Nuclear Translocation 
Dafni-Eleftheria Pefani, Daniela Pankova, Aswin G. Abraham, Anna M. Grawenda, Nikola Vlahov, Simon Scrace, Eric O’ Neill 
Molecular Cell 
Volume 63, Issue 1, Pages (July 2016)
DOI: /j.molcel
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2. Molecular Cell 2016 63, 156-166DOI: (10.1016/j.molcel.2016.05.012)
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3. Figure 1 RASSF1A Is Degraded in Response to TGF-β
(A) Pie chart representing the functional separation of GSEA gene signatures identified in lung and breast tumor cohorts that were split on positive and negative RASSF1A mRNA or high and low promoter methylation (Tables S1 and S2).
(B) Table representing overlapping signatures within each tumor type that were significantly altered in all four analyses. NES, nominal enrichment score; FDR, false discovery rate; p, binomial probability.
(C) Change of RASSF1A levels in HOP92 cells after treatment with TGF-β1 for the indicated times.
(D) Assessment of RASSF1A levels in U2OS TET ON doxycycline-inducible cells (U2OSTetON-RASSF1A) treated with 1μg/ml doxycycline and TGF-β1 for the indicated times.
(E) Assessment of RASSF1A levels in a TGF-β1 time course in the presence or absence of MG132 or E
(F) RASSF1A expression after induction with 1μg/ml doxycycline and treatment with TGF-β1 in the presence or absence of MG132.
(G) U2OS cells expressing FLAG-RASSF1A were transfected with MYC-Ub, treated with TGF-β1, and FLAG-RASSF1A was immunoprecipitated to assess MYC-Ub incorporation. IP, immunoprecipitation.
(H) Western blot analysis of RASSF1A levels in the NBE-1 and MCF10A cell lines after treatment with TGF-β1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
RASSF1A Interacts with TGFβ-RI and Is Targeted by the ITCH E3 Ligase for Degradation
(A) RASSF1A localization in control HOP92 cells (Con) and treated with TGF-β1 for 2 hr. Scale bars, 10 μm.
(B) Co-IP of FLAG-RASSF1A with endogenous TGFβ-RI or TGFβ-RII in the presence or absence of TGF-β1 (2 hr) in U2OS cells.
(C) Graphical representation of RASSF1A mutants used for mapping RASSF1A/TGFβRI interaction and western blot analysis of MYC immunoprecipitation from the indicated inputs from U2OS cells.
(D) Co-IP of ITCH and FLAG-RASSF1A in U2OS cells.
(E) Assessment of RASSF1A levels in HOP92 cells treated with siITCH or siNT after treatment with TGF-β1 for the indicated times.
(F) In vivo ubiquitination assay in U2OS cells transfected with FLAG-RASSF1A to assess MYC-Ub incorporation after treatment with siITCH or control siRNA in response to TGF-β1.
(G) RASSF1A protein levels in HOP92 cells transfected with siITCH and subsequent re-expression of wild-type ITCH (ITCH-WT) or a catalytically inactive mutant (ITCH-CA) in controls and cells treated with TGF-β1 for 2 hr. Quantification was done relative to the control. Data were derived from three experiments, and a representative blot is shown. p values were derived from two-tailed unpaired t test. ∗p < 0.05 was considered significant.
See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
RASSF1A Restricts TGF-β Signaling, Inhibiting YAP1 Binding and Nuclear Accumulation of SMAD2
(A) Co-IP of YAP1 with SMAD2 or p73 in response to increasing concentrations of doxycycline (DOX) and TGF-β1 treatment for 2 hr.
(B) Nuclear SMAD2 in U2OSTetON-RASSF1A upon treatment with doxycycline (1 μg/ml) and stimulation with TGF-β1 for 2 hr. Scale bars, 10 μm.
(C) Quantification of mean SMAD2 nuclear intensity of the cells in (B). Data are presented as mean intensity ± SD from three experiments.
(D) Expression of Smad7, PAI-1, and CTGF determined by qPCR from U2OSTetON-RASSF1A cells treated or not treated with doxycycline (1 μg/ml) and TGFβ-1 for 2 hr. Values and error bars represent the mean ± SD of triplicates and are representative of three independent experiments.
(E) Co-IP between YAP1 and SMAD2 in HOP92 cells treated with control or two different siRNA for RASSF1A and stimulated or not stimulated with TGF-β1 for 2 hr.
(F) Nuclear SMAD2 in Rassf1A+/+ and Rassf1A−/− MEFs after treatment with TGF-β1 for 2 hr. Scale bars, 10 μm. Green outline around the nuclei defines the quantified area.
(G) Percentage of cells with nuclear SMAD2 in the absence of TGF-β1 stimulus.
(H) Nuclear SMAD2 quantification of TGF-β-treated cells from (F). Data are presented as mean intensity ± SD from three experiments.
(I) Expression of Smad7, PAI-1, and CTGF determined by qPCR from HOP92 cells treated with siRASSF1A and induced with TGF-β1 for 2 hr. Data show the mean ± SD of triplicates and are representative of three biological replicates.
p values were derived from two-tailed unpaired t test. ∗p < 0.05 and ∗∗∗p < were considered significant. See also Figures S2 and S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 RASSF1A Inhibits TGF-β-Induced Invasion
(A) Boyden chamber transwell invasion assay of U2OSTetON-RASSF1A cells treated or not treated with TGF-β1 for 24 hr. The bar graph shows the number of invaded cells counted from four random fields.
(B) Relative spheroid invasion in 3D collagen matrix of U2OSTetON-RASSF1A cells in response to TGF-β1 stimulation for 7 hr. Representative images are shown, and quantification of invaded multi-cellular projections (collective) per spheroid was calculated as relative value compared with control cells. Data show the mean ± SD of three experiments.
(C) Boyden chamber transwell invasion assay from siRASSF1A- or control-treated HOP92 cells after being stimulated for 24 hr with TGF-β1. The number of invaded cells and representative images are shown. Doxycycline was used at 1μg/ml for RASSF1A induction. All data represent the mean ± SD and were derived from three independent experiments.
(D) Model of the effect of RASSF1A levels on TGF-β signaling.
p values were derived from two-tailed unpaired t test. ∗p < 0.05 and ∗∗∗p < were considered significant. Scale bars, 200 μm. ns, not significant. See also Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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