Journal: Nature Communications

Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

doi: 10.1038/s41467-017-02786-5

Figure Lengend Snippet: RASSF1A regulates LATS-mediated YAP phosphorylation and enforces differentiation entry via p73. a Subcellular localisation and expression of total YAP and pS127-YAP in gradually differentiating ESC cultures. pS127-YAP is significantly increased upon differentiation. b Size exclusion chromatography of ESC lysates via Gel filtration column and western blotting with indicated antibodies. c Phosphoproteomic analysis of YAP phosphorylation in RASSF1A-expressing versus non-expressing ESC. All cells are transfected with human FLAG–YAP1. All mass-spec intensities were normalised to YAP intensities for each sample. See also Supplementary Fig and Data 4. d Western blotting of YAP immunoprecipitates under indicated conditions for the indicated antibodies and quantification of p73 relative ratio to YAP. e Western blotting of FLAG immunoprecipitates from ESC transfected with the indicated constructs. FLAG–YAP mutants are triple-tagged. f Proximity ligation assay (PLA) demonstrates association of anti-pS127-YAP with anti-p73 antibodies. Red dots indicate positive association. The graph reports number of PLA events between p73/pS127 in the presence or absence of RASSF1A. See also Supplementary Fig . g YAP ChIP on indicated differentiation-related gene promoters and h qPCR for germ layer-specific differentiation markers in ESC in response to indicated conditions. See also Supplementary Data . i ESC cultures expressing zsRASSF1A or empty vector (zsCtrl) were subject to neural differentiation assay via N2B27 and retinoic acid (RA). Scale bars: 25–50 μm. *P < 0.05 , **P < 0.01 and ***P < 0.001 , respectively, of Student’s t -test. Error bars indicate s.e.m. Data shown are representative of at least three independent experiments

Article Snippet: The following primary (dilution 1:100) and secondary (dilution 1:500) antibodies were used: Nanog (ab80892), Oct4 (ab19857), Sox2 (MAB4423), pS127 YAP (Cell Signaling; 4911), p73 (Imgenex;img-246), ki67 (AB9260), YAP (sc-15404 and sc-101199), Nestin (sc-20978), Alexa fluor goat anti-rabbit and anti-mouse (Invitrogen).

Techniques: Phospho-proteomics, Expressing, Size-exclusion Chromatography, Filtration, Western Blot, Transfection, Mass Spectrometry, Construct, Proximity Ligation Assay, Plasmid Preparation, Differentiation Assay

Journal: Nature Communications

Article Title: RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73

doi: 10.1038/s41467-017-02786-5

Figure Lengend Snippet: Premature activation of RASSF1A impairs embryogenesis via p73. a Indicated gene expression levels in published GEO data sets GDS3599 and GDS2156. b Temporal expression of Oct4 and Rassf1A mRNA in the pre-implantation embryo (% of maximum expression) from published GEO data sets GDS752 (black colour) and GDS814 (red colour). c Nuclear localisation of YAP during early stages of pre-implantation development. d Nanog immunofluorescence and e representative images of embryos microinjected with either control (zsCtrl) or RASSF1A-expressing (zsR1A) vectors stained for stem cell marker expression. Bar graph showing total OCT4 protein levels across all embryos in zsR1A versus zsCtrl. f 'Kill curve' to determine lethal RASSF1A concentration in pre-implantation embryos. The graph expresses percentage (%) of blastocyst-forming embryos at the indicated RASSF1A concentration. g Viability of embryos in response to RASSF1A expression and/or sip73 microinjection, n = 15. BF bright field channel. Scale bars: 10–50 μm. *P < 0.05 , of Student’s t -test. Error bars indicate s.e.m

Article Snippet: The following primary (dilution 1:100) and secondary (dilution 1:500) antibodies were used: Nanog (ab80892), Oct4 (ab19857), Sox2 (MAB4423), pS127 YAP (Cell Signaling; 4911), p73 (Imgenex;img-246), ki67 (AB9260), YAP (sc-15404 and sc-101199), Nestin (sc-20978), Alexa fluor goat anti-rabbit and anti-mouse (Invitrogen).

Techniques: Activation Assay, Gene Expression, Expressing, Immunofluorescence, Control, Staining, Marker, Concentration Assay, Microinjection

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Eighty-six genes (brown) were down regulated in the absence of all three p53 family members. A number of genes were down regulated in the absence of each p53 family member individually; 109 in p53 deficient cells (red), 148 in p63 deficient cells (yellow), and 131 in p73 deficient cells (blue). Several genes were down regulated in the absence of two family members; 47 in the absence of p53 and p63 (orange), 58 in the absence of p63 and p73 (green), and 41 in the absence of p53 and p73 (purple).

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Each row represents the specified gene. Each column represents the expression level of a specified knock out MEF line relative to the expression level of wild-type MEFs after DNA damage. The red color indicates upregulation, the green color indicates down regulation, while black indicates no significant change of the indicated gene expression. Clustering based on Euclidean distance indicates that p63- and p73- deficient E1A MEFs are more similar to each other than to p53−/− E1A MEFs. Genes of interest are listed in boxes and are associated with their corresponding location on the heatmap.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Expressing, Knock-Out, Gene Expression

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: p53 family response elements assayed by ChIP.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques:

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Real time PCR analysis of E1A MEFs of the following genotypes (wild-type, p53−/− , p63−/−, p73−/− and p63−/−;p73−/− ) after treatment with (A) doxorubicin (0.34 µM) for 12 hours or (B) γ radiation (12 hours). The Y-axis shows the fold induction. Bars represent 3 MEF lines for each genotype, each performed in triplicate. Data represent the mean ± SEM. The asterisk denotes statistical significance compared to wild-type, p<0.001.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Real-time Polymerase Chain Reaction

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: (A) Western blot analysis for Rad51 using whole cell lysates from wild-type and p63−/−;p73−/− MEFs treated with 0 Gy or 10 min (m), 30 m, 1 hour (h), 2 h and 4 h after 5 Gy of gamma irradiation. Actin was used as a control for equal loading. (B–I) Immunohistochemistry (IHC) of normal mammary tissue or mammary adenocarcinomas from p63+/−;p73+/− mice using antibodies as follows: (B) normal mammary tissue from p63+/−;p73+/− mouse using Rad51 antibody, (C) mammary adenocarcinoma from p63+/−;p73+/− mouse using Rad51 antibody, (D) normal mammary tissue from p63+/−;p73+/− mouse using BRCA2 antibody, (E) mammary adenocarcinoma from p63+/−;p73+/− mouse using BRCA2 antibody, (F) normal mammary tissue from p63+/−;p73+/− mouse using p63 antibody, (G) mammary adenocarcinoma from p63+/−;p73+/− mouse using p63 antibody, (H) normal mammary tissue from p63+/−;p73+/− mouse using p73 antibody, (I) mammary adenocarcinoma from p63+/−;p73+/− mouse using p73 antibody.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Western Blot, Irradiation, Control, Immunohistochemistry

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Chromatin immunoprecipitation (ChIP) analysis using wild-type E1A MEFs (WT) and E1A MEFs deficient for the p53 family members ( p53−/− , p63−/− and p73−/− ) before (U) and after treatment with doxorubicin (D) for 12 hours. Antibodies used to immunoprecipitate protein-DNA complexes in each cell line are shown in various colors: p53 (red), p63 (blue), and p73 (green). Total input chromatin is shown for each sample (input). Each ChIP was performed using 3 independent MEF lines in triplicate.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Chromatin Immunoprecipitation

Journal: PLoS Genetics

Article Title: p63 and p73 Transcriptionally Regulate Genes Involved in DNA Repair

doi: 10.1371/journal.pgen.1000680

Figure Lengend Snippet: Bar graphs showing fold induction for each luciferase reporter gene in (A–D) p63−/−; p73−/− or (E,F) p53−/−;p73−/− primary MEFs. Reporter genes used are as follows: (A,E) pGL3-Rad51-1 containing the binding elements in intron 1, (B) pGL3-Rad51-2 containing the binding elements in intron 2, (C,F) pGL3-BRCA2 containing the binding element in intron 2, and (D) pGL3-mre-11 containing the binding element in intron 1. Pluses above each bar graph indicate which isoforms of p63 or p73 were transfected in cells with the firefly-luciferase reporter genes. Renilla-luciferase was used as a control for transfection efficiency, and pPERP-luc was used as a positive control. Each experiment was performed 6 times using 3 independent MEF lines. Data are represented as the mean ± SEM.

Article Snippet: Cellular proteins were crosslinked to chromatin with 1% formaldehyde. p53-DNA, p63-DNA or p73-DNA complexes were immunoprecipitated using the following antibodies: pan-p63 (4A4, Santa Cruz), pan-p73 (IMG-259a, Imgenex) or p53 (Ab-3, Oncogene Research Products).

Techniques: Luciferase, Binding Assay, Transfection, Control, Positive Control