Journal: bioRxiv

Article Title: Tripartite Inhibition of SRC-WNT-PKC Signalling Consolidates Human Naïve Pluripotency

doi: 10.1101/2020.05.23.112433

Figure Lengend Snippet: a . Schematic and FACS results following using WIBR2 (female 46XX) 29-9 hESC line that carries GFP and tdTomato on each of the X chromosomes in the MECP2 locus. Parental 29-9 clone has the X chromosome carrying mCherry allele in the active state, and thus is positive only for tdTomato and negative for GFP in the primed state. Upon transfer to HENSM conditions, all cells turn on both X chromosomes and thus become double positive for both fluorescent markers (GFP and tdTomato). After transfer into primed conditions (i.e. repriming), cells start to inactivate the X chromosome again. b . RNA-FISH analysis for ATRX transcription in primed and HENSM WIBR3 cells. Note that ATRX is active on both X chromosomes only in HENSM conditions. c . X:Autosome allelic ratios calculated for primed and naïve PSCs (value of each single RNA-seq sample). d. Average methylation as calculated from primed samples, and naïve samples that were maintained in various HENSM conditions, along with previously published Reset-naïve samples (Takashima et al), and human ICM samples . DNMT1 -/- (from TET-OFF lines) samples were used as negative control for methylation. e. Global methylation histogram measured on the same samples as in (d). Dark blue - percentage of highly methylated CpGs (>0.9 methylation level), light blue – percentage of lowly methylated CpGs (<0.1 methylation level). f . Western blot analysis for DNA methylation regulators, DNMT1 and UHRF1 enzymes. DNMT1 protein levels are maintained in all conditions. UHRF1 is partially depleted in HENSM conditions, and this decrease is more enhanced when ERKi concentration is increased. g. Representative images of whole-mount in-toto imaged mouse embryos, after microinjection of the indicated human naïve iPSCs, are shown in comparison to non-injected wild-type embryos. White squares in tiles outline zoomed-in regions in subsequent panels. GFP staining was used to trace hiPSC-derived progeny and CellTracker and Hoechst as counter staining. h. Representative images of IHC for TUJ1 and GFP of injected (upper panels) and non-injected E15.5 mouse embryos (lower panels) for spinal cord region are shown. GFP served as human cell tracer and TUJ1 as neural marker. GFP, TUJ1, overlap as well as merged constitute zoomed-in regions of tissues depicted in red squares in the tiles. White arrowheads in insets depict co-localization of GFP and TUJ1. TUJ1, neuron-specific class III beta-tubulin; GFP, green fluorescent protein; p53, tumor protein p53; iPSC, induced pluripotent stem cell. Tile scale bar 200 and 100µm. Zoomed-in scale bar 50µm.

Article Snippet: Blocking was carried out in 10% skim milk in PBST for 1 h. Blots were incubated with the following antibodies in 5% BSA in PBST: KLF4 (AF3158; 1:200; R&D), OCT4 (H-134; 1:1,000; Santa Cruz), NAANOG (397A; 1:1,000; Bethyl), METTL3 (A301-567A, 1:2000, Bethyl), HSP90beta (ab32568, 1:10000, Abcam), DNMT1 (ab87654, 1:1000, Abcam), GAPDH (ab181602, 1:10000, Abcam), ACTIN (ab6276, 1:10000, Abcam), UHRF1 (sc-373750, 1:1000, Santa Cruz), DGCR8 (10996-1-AP, 1:1000, Proteintech), P53 (D-01, courtesy from Varda Roter’s lab), β-catenin (sc-7963, 1:1000, Santa Cruz), TCF3 (CST2883, 1:1000, Cell Signaling), STAT3 (sc-482, 1:1000, Santa Cruz), KLF4 (sc-20691, 1:1000, Santa Cruz), RBPJ (C5 5313, 1:1000, Cell Signaling), TFAP2C (CSTH2320, 1:1000, Cell Signaling), STAT3 (sc-7993, 1:1000, Santa Cruz).

Techniques: RNA Sequencing, Methylation, Negative Control, Western Blot, DNA Methylation Assay, Concentration Assay, Microinjection, Comparison, Injection, Staining, Derivative Assay, Marker

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer

doi: 10.1073/pnas.0908428107

Figure Lengend Snippet: p53 Drives metastasis of pancreatic ductal adenocarcinoma. (A) Kaplan-Meier survival curve shows no significant difference in survival between Pdx1-Cre-GFP, LSL-KrasG12D, LSL-Trp53R172H/+ (KPC) mice (solid line), and Pdx1-Cre-GFP, LSL-KrasG12D/+, LSL-Trp53loxP/+ (KPflC) mice (dashed line). P = 0.479. (B) Table showing that mean lifespan and median survival are not significantly different in KPC mice compared with KPflC mice, whereas incidence of metastasis in KPC mice is significantly increased compared with KPflC mice, in which metastasis is not observed at all. (C) p53 Histoscore in relation to lymph node status in cases of human PDAC (0, lymph node negative; 1, metastatic disease present in <50% of lymph nodes sampled; 2, metastatic disease present in >50% of lymph nodes sampled; mean number of nodes reviewed per resection, 21). (D and E) H&E-stained sections from (D) a KPC tumor and (E) an age-matched KPflC tumor show there is no difference in tumor stage or grade between the two genotypes. (F and G) H&E-stained sections of liver metastases arising in KPC mice.

Article Snippet: Proteins were transferred to PVDF membrane, blocked, and probed with antibodies against Flag (M2, Sigma), p53 (D01, Pharmingen), and β-tubulin (Sigma).

Techniques: Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer

doi: 10.1073/pnas.0908428107

Figure Lengend Snippet: Senescence program is activated in KrasG12D-expressing cells in the normal pancreas but not in pancreatic tumors. (A–C) β-Galactosidase staining at pH 6, (D–F) p53 immunohistochemical staining, (G–I) p21 immunohistochemical staining, and (J–L) MCM2 immunohistochemical staining, in sections of frozen (β-galactosidase) or formalin-fixed paraffin-embedded pancreatic tissue. (A, D, G, and J) Normal pancreatic ducts in pancreata harvested from 6-week-old Pdx1-Cre-GFP, LSL-KrasG12D/+ (KC) mice, (B, E, H, and K) PanIN lesions in pancreata harvested from 6-week-old KC mice, and (C, F, I, and L) pancreatic ductal adenocarcinoma harvested from Pdx1-Cre-GFP-LSL-KrasG12D, LSL-Trp53R172H (KPC) mice. Arrowheads indicate areas where p21 up-regulation has been lost.

Article Snippet: Proteins were transferred to PVDF membrane, blocked, and probed with antibodies against Flag (M2, Sigma), p53 (D01, Pharmingen), and β-tubulin (Sigma).

Techniques: Expressing, Staining, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer

doi: 10.1073/pnas.0908428107

Figure Lengend Snippet: Mutant p53 promotes invasion of PDAC cells in vitro. Inverted invasion assays were performed on murine PDAC tumor cell lines with or without mutant p53. (A) Tumor cell lines bearing mutant p53R172H, from Pdx1-Cre-GFP, LSL-KrasG12D, LSL-Trp53R172H/+ (KPC) tumors (Middle), invade further than tumor cells grown from Pdx1-Cre-GFP, LSL-KrasG12D/+, LSL-Trp53loxP/+ (KPflC) tumors (Top). Introduction of exogenous expression of mutant p53R175H into these KPflC tumor cells, however, promotes invasion (Bottom). (B) Bar graph showing increased invasive capacity of KPC tumor cells compared with KPflC tumor cells. Representative images of at least three independent experiments are shown. Columns indicate mean; bars indicate SE. *P ≤ 0.01. (C) Western immunoblotting shows expression of flag-tagged p53R175H in stably transfected KPflC cells. β-Tubulin represents a loading control. (D) Bar graph showing increased invasive capacity of KPflC tumor cells following exogenous expression of mutant p53R175H. Representative images of at least three independent experiments are shown. Columns indicate mean; bars indicate SE. *P < 0.01 by unpaired Student’s t test.

Article Snippet: Proteins were transferred to PVDF membrane, blocked, and probed with antibodies against Flag (M2, Sigma), p53 (D01, Pharmingen), and β-tubulin (Sigma).

Techniques: Mutagenesis, In Vitro, Expressing, Western Blot, Stable Transfection, Transfection

Journal: Cancer Research

Article Title: Cells with Defective p53-p21-pRb Pathway Are Susceptible to Apoptosis Induced by p84N5 via Caspase-6

doi: 10.1158/0008-5472.can-07-0334

Figure Lengend Snippet: Figure 1. A, analyses of uninfected cells (control) and cells infected with AAV at various times after infection. Cellular DNA was stained with propidium iodide and analyzed by FACS. B, Western blot analysis of activated p53 (p53 Ser15P) and p21 from lysates of uninfected U2OS cells and AAV-infected cells at various days post infection. C, Western blot analyses of lysates of Saos-2 cells (c) that were transduced with either empty vector (v) or vector that carried a p21 gene (+p21). D, FACS analyses of DNA content and methylene blue staining of uninfected cells and AAV-infected untransduced and transduced cells.

Article Snippet: Equal amounts of protein were loaded onto SDSpolyacrylamide gels and transferred onto nitrocellulose membranes and analyzed with antibodies to p53 (Novocastra, NCL-p53-D01), phospho-Ser15 p53 (Cell Signaling), p21 (Santa Cruz Biotechnology), pRb (BD PharMingen), actin (Santa Cruz Biotechnology), caspase-3 and caspase-6 (Cell Signaling), or p84N5 (BD Transduction).

Techniques: Control, Infection, Staining, Western Blot, Transduction, Plasmid Preparation

Journal: Cancer Research

Article Title: Cells with Defective p53-p21-pRb Pathway Are Susceptible to Apoptosis Induced by p84N5 via Caspase-6

doi: 10.1158/0008-5472.can-07-0334

Figure Lengend Snippet: Figure 3. A, Western blot analysis of lysates from cells that were transduced with retroviruses encoding either scrambled shRNA (shC) or pRb-specific shRNA (shRb). Antibodies against pRb and p53 were used. On extensive exposure of the blot, a residual pRb protein band is observed in the shRb lane, indicating noncomplete abolition of the pRb protein by the shRNA. B, methylene blue staining of AAV-infected cells transduced with scrambled shRNA or pRb-specific shRNA.

Article Snippet: Equal amounts of protein were loaded onto SDSpolyacrylamide gels and transferred onto nitrocellulose membranes and analyzed with antibodies to p53 (Novocastra, NCL-p53-D01), phospho-Ser15 p53 (Cell Signaling), p21 (Santa Cruz Biotechnology), pRb (BD PharMingen), actin (Santa Cruz Biotechnology), caspase-3 and caspase-6 (Cell Signaling), or p84N5 (BD Transduction).

Techniques: Western Blot, Transduction, shRNA, Staining, Infection

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A, D, G and J ) mRNA expression of p53, Mdm2, p21 and Bax respectively, in placental lysates at delivery. ( B, E, H, and K ) Protein expression of p53, Mdm2, p21 and Bax, respectively, through densitometry standardized to Myosin Light Chain (MLC) (*p<0.05, **p<0.01, n = 8). ( C, F, I, and L ) Localisation of p53, Mdm2, p21 and Bax respectively, in placental villi by immunohistochemistry. ST = Syncytiotrophoblast. Images counterstained with haematoxylin. Scale bar = 5 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Expressing, Immunohistochemistry

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Transfection of placental explants with fluorescent labelled non-silencing siRNA demonstrating the presence of siRNA in cytotrophoblast (CT) and syncytiotrophoblast (ST). ( B and C ) Quantitative PCR demonstrating significant reductions in p53 and Mdm2 respectively, in explants treated with p53 or Mdm2 siRNA (*p< 0.05, **p<0.01, n = 6). ( D and E ) Western blot densitometry and immunohistochemistry showing a reduction in Mdm2 protein following treatment with Mdm2 siRNA. p53 expression is increased in trophoblast cytoplasm and nuclei in response to Mdm2 siRNA. ( F and G ) TUNEL index and images respectively, showing apoptosis significantly increased in explants exposed to Mdm2 siRNA (*p<0.05). Elevated apoptosis confirmed by M30 immunostaining with examples imaged by electron microscopy. ( H ) Proliferative index and representative images showing significantly reduced proliferation in explants cultured with Mdm2 siRNA (*p<0.05). ( I ) density of syncytial nuclear aggregates (SNA) increased by treatment Mdm2 siRNA (*p<0.05), representative images of control and Mdm2 siRNA (arrow = SNA). Representative images shown (FC = fetal capillary, IVS = intervillous space, CT = cytotrophoblast, ST = syncytiotrophoblast). All scale bars = 10 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Expressing, TUNEL Assay, Immunostaining, Electron Microscopy, Cell Culture

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Representative Western Blots of control and Nutlin-3 treated normal placental lysates. Densitometry revealed a significant increase in the expression of ( B ) p53, ( C ) Mdm2, ( D ) p21, ( E ) Puma (*p<0.05, n = 5). ( F ) There was no effect on Bax. Co-treatment of BeWo cells with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) reduced caspase-3/7 activity ( G ) and TUNEL staining ( H ) to the level of controls. Representative images of TUNEL staining in ( I ) control, ( J ) Nutlin-3 and ( K ) co-treatment with Nutlin-3 and Pifithrin-α. Blue = DAPI, Green = TUNEL. Scale bar = 50 µm.

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Western Blot, Expressing, Activity Assay, TUNEL Assay, Staining

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: ( A ) Western Blots of Nutlin-3 treated BeWo cell lysates and co-treatment with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) demonstrated no effect upon ( B ) p53, ( C ) Mdm2, ( D ) p21, ( E ) Puma or ( F ) Bax protein expression (n = 5).

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: Preeclampsia Is Associated with Alterations in the p53-Pathway in Villous Trophoblast

doi: 10.1371/journal.pone.0087621

Figure Lengend Snippet: Co-treatment with Nutlin-3 (30 µM) and Pifithrin-α (10 µM) had no effect on ( A ) p53, ( B ) Mdm2 or ( E ) Bax mRNA expression. Treatment with Nutlin-3 increased ( C ) p21 and ( D ) Puma expression an effect lost by co-treatment with Pifithrin-α (*p<0.05, **p<0.01, n = 5).

Article Snippet: Membranes were blocked for 1 hr with 3% (w/v) milk in Tris-buffered saline containing 0.05% (v/v) Tween-20 (TBS-T) and then probed overnight at 4°C, with mouse monoclonal antibodies to either p53 (Clone D01, Merck Biosciences, Nottingham, UK, 1 µg/ml (Explants), 0.1 µg/ml (BeWo)), Mdm2 (Clone 2A10, Merck Biosciences, 2 µg/ml), anti-p21 (Clone EA10, Merck Biosciences, 1∶100), Bak (TC-102, Merck Biosciences, 1∶200), Bcl-2 (Clone 100/D5, Abcam, Cambridge, UK, 1 µg/ml), Procaspase-3 (Clone 84803, R&D Systems, Abingdon, UK, 1∶1000), Procaspase 8 (Clone 84131, Merck Biosciences, 1∶100), Myosin Light Chain (Clone MY21, Abcam, 0.1 µg/ml), β-actin, (Clone AC15, Sigma, 1∶10,000) or rabbit polyclonal antibody against Bax (ab7977, Abcam, 1 µg/ml (Explants), 0.2 µg/ml (BeWo)), p21 (Abcam, 0.2 µg/ml (BeWo)), Puma (Abcam, 4 µg/ml) or β-actin (Clone AC15, Sigma, 1∶10,000).

Techniques: Expressing