Journal: International journal of oncology

Article Title: Epithelial membrane protein 3 regulates lung cancer stem cells via the TGF‑β signaling pathway.

doi: 10.3892/ijo.2021.5261

Figure Lengend Snippet: Figure 2. EMP3 regulates CSC properties in lung CSC. (A) Western blot analysis of the CSC marker proteins CD133, ALDH1A1, ALDH1A3 and CD44. The CSC regulatory proteins Sox2, Oct‑4 and Nanog were also analyzed by western blotting. A549 cells were transfected with siRNA targeting EMP3. (B) Immunocytochemistry analysis of CSC marker proteins using siRNA treated A549 cells. The green signal is produced by the GFP tag on the secondary antibody. (C) Sphere‑forming capacity analysis of A549 cells in siRNA transfected EMP3 cells and (D) the ability to of an anti‑EMP3 antibody to abrogate sphere formation was assessed. (E) Single‑cell assay of si‑EMP3 transfected cells and (F) the ability of the anti‑EMP3 antibody to abrogate this. (G) Limiting dilution assays were performed in 96 well plates. Wells were plated with 1, 50, 100, 150 or 200 cells/well, and conditioned media was added. Results were confirmed 10 days after seeding of cells. Colony‑formation assays to observe the clonogenicity of the (H) EMP3‑knockdown A549 and (I) anti‑EMP3 antibody treated A549 cells. Cells were irradiated with 3 Gy radiation. After 10 days of incubation, the colonies were stained with crystal violet. (J) Western blot analysis of members of the Sonic hedgehog, Wnt/β‑catenin and Notch signaling pathways in CSCs. Data are presented as the mean ± standard deviation of three repeats. Scale bar, 50 µm. *P<0.05 vs. control. EMP3, epithelial membrane protein 3; ALDH1, aldehyde dehydrogenase 1; CSC, cancer stem cell; Sox2, sex determining region Y‑box 2; Oct‑4, octamer‑binding transcription factor 4; siRNA, small interfering RNA; p, phosphorylated; GSK3‑β, glycogen synthase kinase 3‑β.

Article Snippet: Antibodies against EMP3 (cat. no. ab236671; Abcam), Sox2 (sex determining region Y‐box 2; cat. no. 3579; Cell Signaling Technology, Inc.), phos‐ phorylated (p)‐Smad2 (cat. no. 18338; Cell Signaling Technology, Inc.), p‐Smad3 (cat. no. 9520; Cel l Signaling Technology, Inc.), Smad2/3 (cat. no. 5678; Cell Signaling Technology, Inc.), β‐actin (cat. no. sc‐7963; Santa Cruz Biotechnology, Inc.), TGFBR1 (cat. no. sc‐518086; Santa Cruz Biotechnology, Inc.), TGFBR2 (cat. no. sc‐17791; Santa Cruz Biotechnology, Inc.), TGFBR3 (cat. no. sc‐74511; Santa Cruz Biotechnology, Inc.), CD44 (cat. no. 5640; Cell Signaling Technology, Inc.), β‐catenin (cat. no. sc‐7963; Santa Cruz Biotechnology, Inc.), Twist (cat. no. sc‐15393; Santa Cruz Biotechnology, Inc.), Vimentin (MA5‐16409; Thermo Fisher Scientific, Inc.), alde‐ hyde dehydrogenase (ALDH1)A1 (cat. no. ab52492; Abcam), ALDH1A3 (cat. no. ab129815; Abcam), Snail (cat. no. sc‐10432; Santa Cruz Biotechnology, Inc.), Slug (cat. no. sc‐166476; Santa Cruz Biotechnology, Inc.), ZEB1 (Zinc finger E‐box‐binding homeobox 1; cat. no. sc‐25388; Santa Cruz Biotechnology, Inc.), E‐cadherin (cat. no ab15148; Abcam), N‐cadherin (cat. no. 610921; BD Biosciences) and Oct4 (cat. no. 2750; Cell Signaling Technology, Inc.) were used.

Techniques: Western Blot, Marker, Transfection, Immunocytochemistry, Produced, Irradiation, Incubation, Staining, Protein-Protein interactions, Standard Deviation, Control, Membrane, Small Interfering RNA

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Targeting CRABP-II overcomes pancreatic cancer drug resistance by reversing lipid raft cholesterol accumulation and AKT survival signaling

doi: 10.1186/s13046-022-02261-0

Figure Lengend Snippet: CRABP-II regulates cholesterol metabolic genes expression through cooperation with HuR. ( A ) Molecular and cellular function analysis by IPA software (Qiagen) based on gene expression microarray profiling. The altered lipid synthesis and accumulation functions upon CRABP-II knockout were listed. ( B ) Heat map of altered cholesterol metabolic genes. ( C, D, E ) Cholesterol metabolic genes expression assessed by Q-PCR. ( F ) Correlation between cholesterol metabolic genes and CRABP-II expression in human pancreatic cancer specimens by Pearson’s product-moment correlation coefficient analysis (PPMCC). Data shown here are combination of Pei Pancreas and Badea Pancrease datasets ( n = 75) from Oncomine. ( G ) Interaction between CRABP-II and HuR identified by co-immuprecipitation (co-IP). GR4000 cell lysis was incubated with anti-CRABP-II rabbit polyclonal antibody and the pull down proteins were separated and blotted with anti-HuR mouse monoclonal antibody. ( H ) Half-life of SREBP-1c mRNA assessed by actinomycin D treatment following with Q-PCR. ( I ) RNA-immunoprecipitation (RIP). The down pulled SREBP-1c mRNA from flagged-CRABP-II transfected CIIKO cells and empty vector transfected cells were assessed by Q-PCR. The actin mRNA was used as control. The experiment was repeated three times and the error bars present standard deviation (SD). **, p < 0.01

Article Snippet: Antibodies used in this study include: CRABP-II mouse mAbs (Millipore, MAB5488), CRABP-II rabbit polyclonal antibody (Proteintech, 10,225–1-AP), HuR (3A2, Santa Cruz, sc-5261), Flotilin-2 (Santa Cruz, sc-28320), GAPDH (Santa Cruz, sc-365062), and Actin (Santa Cruz, sc-1615), anti-Flag M2 mAb (Sigma, F9291), anti-Flag agarose beads (Clontech, #635,686), Ki67 (SP6, ThermoFisher, RM-9106-S0), ADRP (Novus, NB110-40,877), Caspas3 (Cell Signaling, #9662), PARP (Cell Signaling, #9542), AKT (Cell Signaling, #4691), mTOR (Cell Signaling, #2983), S6 (Cell Signaling, #2217), pAKT (S473, Cell Signaling, #9018), pmTOR (Cell Signaling, #5536), pS6 (Cell Signaling, #4858), and pGSK3β (Cell Signaling, #5558).

Techniques: Expressing, Cell Function Assay, Software, Gene Expression, Microarray, Knock-Out, Co-Immunoprecipitation Assay, Lysis, Incubation, RNA Immunoprecipitation, Transfection, Plasmid Preparation, Control, Standard Deviation

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Intact protein LC–MS spectra of Pin1 (black) directly identify covalent binders (blue) in the electrophilic library screen (200 μM compound for 24 h). Madduct indicates the mass of the expected adduct for the indicated example. b, Distribution of hits in the Pin1 screening campaign and their corresponding labeling (%). Nine hits (18.75%) out of the 48 top hits that labeled Pin1 at >75% (dark and light blue) share sulfolene or sulfolane moieties. Labeling percentage calculated as previously described28. c, 2D analysis of the top ten optimized binders (structures shown in f); labeling percentage in the LC–MS assay plotted against reactivity (log (K)) suggests Sulfopin for further biological evaluation. d, Fluorescence polarization assay with the top ten binders, including juglone and a nonreactive control (Sulfopin-AcA), after 14 h of preincubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. See Supplementary Table 3 for apparent Ki. mP represents the polarization value. e, PPIase substrate activity assay of Pin1 with Sulfopin (n = 3) and juglone (n = 2). Data points are plotted as the average of independent experiments ± s.e.m. for Sulfopin. f, Structures of the top ten binders in the Pin1-labeling LC–MS assay, the nonreactive control Sulfopin-AcA and juglone. g, X-ray crystal structure of Pin1 in complex with Sulfopin (1.4-Å resolution, PDB code 6VAJ). Pin1 (white) with relevant side chains in stick representation; Sulfopin is shown in pink. Hydrogen bonds are depicted as dashed lines. AU, arbitrary units.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Liquid Chromatography with Mass Spectroscopy, Labeling, Fluorescence, Activity Assay

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Fluorescence polarization assay showing that the DTB-labeled probe, Sulfopin-DTB, binds Pin1 with similar potency to Sulfopin following 14 h of incubation with Pin1. Data points are plotted as the average of n = 3 independent samples ± s.e.m., and are representative of n = 2 independent experiments. b, Chemical structure of Sulfopin-DTB. c, Sulfopin shows time-dependent engagement in PATU-8988T cells. PATU-8988T cells were treated with Sulfopin (1 μM) for the indicated time points followed by cell lysis, incubation with Sulfopin-DTB (1 μM), streptavidin pulldown and immunoblot analysis. d,e, Sulfopin shows long-term engagement of Pin1. PATU-8988T (d) or HCT116 (e) cells were incubated with or without Sulfopin for the indicated time points, followed by cell lysis, incubation with DTB probe, streptavidin pulldown and immunoblot analysis. Substantial engagement (>50%) was still evident after 72 h. f,g, Sulfopin fully engages Pin1 in PATU-8988T cells at 1 μM and in HCT116 cells at 0.5 μM (see Supplementary Fig. 9b for the structure of BJP-DTB). PATU-8988T (f) or HCT116 (g) cells were incubated with Sulfopin at the indicated concentrations for 5 h, followed by cell lysis, DTB probe incubation (1 h, 1 μM), streptavidin pulldown and immunoblot analysis. The noncovalent control, Sulfopin-AcA, is unable to outcompete Pin1 pulldown. c–g, Results are representative of n = 2 independent experiments. h, Sulfopin engages Pin1 in vivo. Mice were treated by oral gavage with the indicated amounts of Pin1 over 2 days for a total of three doses. Following this treatment, spleens were lysed for a competition pulldown experiment with Sulfopin-DTB. Results are representative of n = 2 independent pulldown experiments, starting from the same spleen lysates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Fluorescence, Labeling, Incubation, Lysis, Western Blot, In Vivo

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, CITe-Id profiling results showing Sulfopin-DTB-labeled cysteine sites, rank ordered by competitive dose response to Sulfopin. Out of 162 cysteine residues reproducibly labeled by Sulfopin-DTB in n = 2 independent experiments, Pin1 C113 was the only site identified with a competitive dose response >2 s.d. from the mean value of the null. (see Supplementary Dataset 3a for a full list of identified peptides, and Supplementary Fig. 10 for results with 12/24-h treatment). b, Waterfall plot showing competitive dose dependency of Pin1 C113 labeling in the CITe-Id experiment. Bars represent mean of n = 2 independent experiments. c, Out of 2,134 cysteines identified in the rdTOP-ABPP experiment, only two showed a light/heavy ratio of >2.5 and, of these, one did not replicate and only Pin1 C113 showed the maximal ratio of 15 in both replicates.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Labeling

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, HeLa cells were treated with either DMSO, Sulfopin, or Go6976 (a Chk1 inhibitor) and exposed to 7.5 Gy IR 1 h after drug treatment. Viability was assessed 3 days post-IR. Sulfopin shows a dose dependent sensitization of the cells to irradiation (n=3; data are represented as mean values with standard deviation). b, Western blot analysis was performed 24 h post-IR, showing Sulfopin blocked phosphorylation of Thr209 of IRAK1. c, A shorter exposure shows that Sulfopin inhibits IRAK1 phosphorylation already at concentrations of 0.1 μM. d, A scheme for testing the effect of Sulfopin in vivo on germinal center B cells in response to immunization. e, Representative flow cytometric plots with Vehicle and Sulfopin (left) and quantification (right) of FASHi CD38− germinal center (GC) cells in WT mice 11 days after immunization with NP-OVA. ** p<0.01, two tailed Student’s t test.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Knock-Out, Irradiation, Standard Deviation, Western Blot, In Vivo, Two Tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, We previously27 generated a PATU-8988T Pin1 knockout (KO) cell line (Supplementary Fig. 12a). Sulfopin (1 μM) had a significant effect on cellular viability after 6 and 8 days (P = 0.01 and P = 0.01, respectively) in WT PATU-8988T cells (left), but showed no significant effect on viability in Pin1 KO cells (right); day 0-normalized growth rate for n = 3 biologically independent samples. b, Relative viability of PATU-8988T WT and Pin1 KO cells grown in 100% Matrigel domes following treatment with either Sulfopin (1 μM; n = 9 biologically independent samples; P = 1.24 × 10−18) or the noncovalent negative control, Sulfopin-AcA (1 μM; n = 9 biologically independent samples). Sulfopin-AcA showed no effect in any of the tested systems. c, Proportion of cells in various cell cycle stages as a function of Sulfopin treatment. The viability effects of Sulfopin are mediated by delayed cell cycle. PATU-8988T cells were treated with either DMSO, 2.5 μM Sulfopin or Sulfopin-AcA for 4 days. Cell cycle analysis was performed by BrdU and propidium iodide staining, followed by FACS analysis. Sulfopin treatment reduced the percentage of cells in S phase (P = 0.0004) and, in turn, increased the number of cells found in G1 phase (P = 0.003), while the noncovalent Sulfopin-AcA did not show this effect (n = 4; see Extended Data Fig. 3 for representative FACS analysis graphs and quantification of the results from two independent experiments). d, Cell culture growth curves. Sulfopin showed variation in antiproliferative effects across cancer cell lines Kuramochi, MDA-MB-468, NGP and NBL-S, with the most pronounced sensitivity observed in MDA-MB-468 cells (day 0-normalized growth rate for n = 3 biologically independent samples; P values for 2.5 μM Sulfopin after 4, 6 and 8 days were 0.007, 0.004 and 0.0004, respectively). Importantly we noted significant viability effects in Myc-high neuroblastoma cell lines NGP and NBL-S (P = 0.018 and 0.002, respectively for 2.5 μM Sulfopin after 8 days). Data points were plotted as the average of n = 3 biologically independent samples ± s.e.m. Statistical significance for all panels was calculated using one-tailed Student’s t-test with unequal variance (NS, not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Generated, Knock-Out, Negative Control, Cell Cycle Assay, Staining, Cell Culture, One-tailed Test

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, PATU-8988T cells were treated for 5 or 6 days with either DMSO (0.1%), Sulfopin (1 μM, 2.5 μM) or the non-covalent control Sulfopin-AcA (2.5 μM). The cells were lysed and activation of caspase 3 and Pin1 levels were analysed by Western blot. As a positive control for caspase 3 activation the cells were treated with Staurosporin (1 μM, 4h; STS). See Supplementary Fig. 13a for the results of an additional independent experiment. Caspase 3 was not activated and Pin1 levels were not changed by the treatment with Sulfopin. b, PATU-8988T cells were treated in triplicates for 6 days with either DMSO (0.1%), Sulfopin (1 M, 2.5 M) or the non-covalent control Sulfopin-AcA (2.5 M). The cells were then stained with AnnexinV-FITC/ 7AAD and analysed by FACS. Staurosporin treatment (1 M, 4h) was used as a positive control for apoptosis. Representative FACS analysis graphs and a quantification of the results (n=3; data are represented as mean values with standard deviation). See Supplementary Fig. 13b for the results of an additional independent experiment. Live cells were defined as AnnexinV−/7AAD−, early apoptosis AnnexinV+/7AAD− and late apoptosis AnnexinV+/7AAD+.

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: Activation Assay, Western Blot, Positive Control, Staining, Standard Deviation

Journal: Nature chemical biology

Article Title: Sulfopin is a covalent inhibitor of Pin1 that blocks Myc-driven tumors in vivo

doi: 10.1038/s41589-021-00786-7

Figure Lengend Snippet: a, Results of an RNA-seq experiment comparing changes in RNA levels between Mino B cells treated with either Sulfopin (1 μM, 6 h, in triplicate) or DMSO. Each dot represents log2 fold change of a transcript (x axis) versus the P value for significance of that change (y axis; Wald test, as implemented in DESeq2). The dotted line indicates P = 0.05; 206 genes were significantly downregulated. b, Results of gene set enrichment analysis using Enrichr against the ENCODE TF chromatin immunoprecipitation–sequencing set. Two of the sets most enriched were Myc target genes from different cell lines. c, HEK293 cells were transfected with 4× E-box luciferase reporter for Myc transcriptional activity levels. Cotransfection with Pin1 increased reporter activity, while 48-h treatment with Sulfopin significantly (one-tailed Student’s t-test) reduced this activity compared to DMSO (n = 3; error bars indicate s.d.).

Article Snippet: The membrane was blocked using 5% BSA in TBST (w/v) for 1 h at room temperature, washed 3× for 5 min with TBST and incubated with the following primary antibodies: cleaved caspase 3 (Cell Signaling, catalog no. 9661, 1:500, overnight at 4 °C), Pin1 (Cell Signaling, catalog no. 3722, 1:1,000, overnight at 4 °C) and β-actin (Cell Signaling, catalog no. 3700, 1:1,000, 1 h at room temperature).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Transfection, Luciferase, Activity Assay, Cotransfection, One-tailed Test

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Schematic representation of the mouse Suv39h2 gene locus and domain structure of the Suv39h1 and Suv39h2 enzymes showing the N-terminal basic domain of Suv39h2 in yellow. ( B ) Western blot of chromatin extracts from wild type and Suv39h dn mouse ES cells (ESC) and fibroblasts (iMEF) to detect endogenous Suv39h1 (48 kDa) and Suv39h2 (53 kDa). An antibody specific for the basic domain of Suv39h2 also detects endogenous Suv39h2 at 53 kDa in wild type but not in Suv39h dn chromatin extracts. The asterisks indicate nonspecific bands. ( C ) Generation of rescued Suv39h dn mouse ES cell lines that express the indicated Suv39h-EGFP constructs under the control of a β-actin promoter. ( D ) Western blot of whole cell extracts from unsynchronized and nocodazole-synchronized mouse ES cell lines to examine expression of the various EGFP-tagged Suv39h products with an α-GFP antibody or with α-Suv39h1 and α-Suv39h2 antibodies to compare their expression levels with regard to the endogenous Suv39h1 and Suv39h2 proteins. H3K9me3 and H3S10phos levels were also analyzed. Histone H3 and Gapdh served as loading controls. DOI: http://dx.doi.org/10.7554/eLife.25293.002

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Western Blot, Construct, Control, Expressing

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Schematic representation of the domain structure of the Suv39h2 enzyme highlighting the N-terminal basic domain (encoded by exon 1) in yellow. The amino acid sequence of this basic domain (aa 1–81) contains 19 arginines (R) and 3 lysines (K) denoted by orange circles. The underlined amino acids correspond to the sequence of the double-branched peptide used for the generation of rabbit polyclonal α-Suv39h2-basicD antibodies. ( B ) Western blot analysis of cytoplasmic (C), nucleoplasmic (N) and chromatin extracts (Chr) from mouse ES cells (ESC) using the α-Suv39h2-basicD antibody (Diagenode #A1438) indicates the presence of endogenous Suv39h2 (~53 kDa) in chromatin extracts of wild-type (wt26) but not in Suv39h dn (dn57) mutant ESC (middle panel). Immunoblotting of GST-Suv39h1 and GST-Suv39h2 recombinant proteins with the α-Suv39h2-basicD antibody (Diagenode #A1438) shows the specific recognition of full-length GST-Suv39h2 (~80 kDa) but not of GST-Suv39h1 (right panel). The asterisks indicate nonspecific bands. ( C ) The sequence of the basic domain of mouse Suv39h2, spanning amino acids 1–81, was queried using UniProt BLAST against a preselected mouse Swiss-Prot database that was filtered with the Entrez query ‘arginine rich’. The top ten hits from this query are listed and proteins with a described RNA binding activity are indicated in bold. Also shown are amino acid sequence alignments of the basic domain of Suv39h2 with two splicing factors (Srsf4 and Sfr19) and one RNA helicase (Ythdc2). Conserved arginine residues are highlighted in yellow and other conserved residues in light blue. DOI: http://dx.doi.org/10.7554/eLife.25293.003

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Sequencing, Western Blot, Mutagenesis, Recombinant, RNA Binding Assay, Activity Assay

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Immunofluorescence analysis for the various Suv39h-EGFP products and for H3K9me3 in interphase nuclei of the rescued mouse ES cell lines. The heterochromatic foci were counterstained with DAPI. Scale bar, 10 μm. ( B ) Reverse transcription quantitative PCR (RT-qPCR) with total RNA isolated from unsynchronized wild type, Suv39h dn or Suv39h-EGFP rescued mouse ES cell lines to detect expression from the major satellite repeats with MSR-specific primers. The amplified signals were normalized to Gapdh and are plotted in the histogram. The data represent the mean ± SD of at least two independent experiments. ( C ) Directed ChIP for H3K9me3 at the major satellite repeats in unsynchronized wild type, Suv39h dn or Suv39h-EGFP rescued mouse ES cell lines. The data represent the mean ± SD of at least two independent experiments. ( D ) Confocal spinning disc immunofluorescence analysis for the various Suv39h-EGFP products and for H3K9me3 at mitotic chromosomes that were presented in nocodazole-synchronized mouse ES cells. For each image, between 30–50 nuclei displaying mitotic chromosomes were analyzed. Scale bar, 10 μm. ( E ) Enlarged images of representative confocal IF analyses of mitotic chromosomes as described in ( D ). Only DAPI and EGFP signals are shown. Scale bar, 10 μm. ( F ) RT-qPCR as described in ( B ), but with total RNA preparations from nocodazole-synchronized mouse ES cells. ( G ) Directed ChIP for H3K9me3 as described in ( C ), but with chromatin material from nocodazole-synchronized mouse ES cells. ( H ) Chromatin release assay for endogenous Suv39h1 (wild type) and Suv39h1-EGFP in rescued mouse ES cells. Proteins were detected by Western blot in the soluble (S) or pellet (P) fractions after progressive (0, 2, 4, 6, 8 and 10 min.) MNase digestion of chromatin from nocodazole-synchronized mouse ES cells. Intensity of protein bands in the S or P fraction was quantified by ImageJ software. Chromatin release is measured by the S/P ratio, which is plotted in the indicated graph. ( I ) FACS profile (propidium iodide labeling) of nocodazole-synchronized wild-type mouse ES cells. ( J ) Chromatin release assay for endogenous Suv39h2 (wild type), Suv39h2-EGFP and the Suv39h-D(T3K81)-EGFP mutant as described in ( H ). DOI: http://dx.doi.org/10.7554/eLife.25293.004

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Isolation, Expressing, Amplification, Release Assay, Western Blot, Software, Labeling, Mutagenesis

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: Total RNA isolated from wild type, Suv39h double null and Suv39h double null mouse ES cells rescued with EGFP-tagged Suv39h1, Suv39h2 or Suv39h2-D(T3K81) was used for reverse transcription quantitative PCR (RT-qPCR). The transcriptional output of LINE L1MdA-5’ UTR repeats was analyzed in unsynchronized ( A ) and nocodazole-arrested cells ( B ). The amplified signal was normalized to Gapdh and the results were plotted relative to the wild type signal. Error bars represent the standard deviation calculated from two biological replicates. DOI: http://dx.doi.org/10.7554/eLife.25293.005

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Standard Deviation

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Workflow of the chromatin release assay in unsynchronized mouse ES cells. Whole cell lysates from mouse ES cells were digested with 10 U of MNase for different time points (0,2,4,6,8 and 10 min) and proteins from the soluble and insoluble chromatin fractions were detected by western blotting. ( B ) Chromatin release for endogenous Suv39h1 in wt ES cells and for Suv39h1-EGFP in rescued ES cells. Protein bands in the soluble fraction (S) or in the pellet (P) were quantified by ImageJ software. Chromatin release is measured by the S/P ratio, which is plotted in the indicated graph. ( C ) Chromatin release assay for endogenous Suv39h2 in wt ES cells and for Suv39h2-EGFP and the Suv39h2-D(T3K81)-EGFP mutant in rescued ES cells as described in ( B ). DOI: http://dx.doi.org/10.7554/eLife.25293.006

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Release Assay, Western Blot, Software, Mutagenesis

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Schematic representation of full-length Suv39h1, full-length Suv39h2, the Suv39h2ΔBD mutant and the Suv39h2-basicD(1-117) proteins. These were expressed as recombinant 6xHisMBP-fusion proteins and their purity is visualized by Coomassie staining. ( B ) Electrophoretic mobility shift assays (EMSA) with the indicated recombinant 6xHisMBP-Suv39h products and 3'-Cy5-labeled in vitro transcribed full-length (234 nt) ss-forward or ss-reverse MSR transcripts (upper panel) or with 3'-Cy5-labeled in vitro transcribed full-length (208 nt) ss-forward or ss-reverse LINE 5'UTR transcripts (lower panel). K D values that are within the tested protein concentration range of 16 nM to 2 μM were calculated with GraphPad Prism6 software and are indicated in the white boxes. The same amount (50 nM) of IVT MSR or LINE 5'UTR transcripts was used although there was reduced 3'-Cy5 labeling efficiency with the LINE 5'UTR ssRNA. ( C ) EMSA with recombinant GST-Suv39h2-basicD(1-117) and 5’-Cy5-labeled DNA or RNA oligonucleotides (35 nt each) from subunit 2 of the MSR that are probed as single-stranded, double-stranded or as RNA:DNA hybrid binding substrates. ( D ) Same EMSA as in ( C ), but with 5’-Cy5-labeled ssRNA oligonucleotides (35 nt) from an Oct4P4 lnc RNA , the MSR reverse RNA and a SINE B1 reverse RNA. For comparison, this EMSA was also done with recombinant GST-HP1α. DOI: http://dx.doi.org/10.7554/eLife.25293.007

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Mutagenesis, Recombinant, Staining, Electrophoretic Mobility Shift Assay, Labeling, In Vitro, Protein Concentration, Software, Binding Assay, Comparison

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Sucrose gradient fractionation of MNase-digested chromatin from the Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2-D(T3K81)-EGFP mouse ES cell lines. The separation between nucleosome-free and nucleosome-containing fractions was monitored by Gelred staining of DNA. The sedimentation profile of the various Suv39h-EGFP products and of endogenous Dnmt3a, HP1α and of H3K9me3 was analyzed by western blotting with α-GFP, α-Dnmt3a, α-HP1α and α-H3K9me3 antibodies (first panel). To address whether RNA is associated with the nucleosomal fractions, MNase-digested soluble chromatin was incubated with RNaseH (second panel) or with RNaseA at 350 mM salt (third panel) or with RNaseA at 100 mM salt (fourth panel) before being loaded on the sucrose gradient. The asterisk indicates unspecific bands. All of these experiments were performed with two biological replicates and the RNaseA (100 mM salt) and RNaseH treatments were done three independent times. ( B ) Gelred DNA staining of sucrose gradient fractionation of MNase-digested chromatin from wild type and Suv39h dn ES cells and RT-qPCR to detect MSR and LINE L1MdA 5'UTR transcripts in RNA preparations from every second fraction of the sucrose gradient. No signal was detected in the control reactions lacking RT. The histogram on the left shows expression of MSR and LINE L1 MdA transcripts in nuclear RNA of wild-type and Suv39h dn ES cells. ( C ) Hiseq RNA sequencing of chromatin-associated and nucleoplasmic cDNA libraries (non-poly(A) selected) that were generated from wild-type ES cells to quantify the relative abundance of minor satellite repeat, major satellite repeat, LINE L1MdA and SINE B1 transcripts. Plotted are the mean counts of three biological replicates. DOI: http://dx.doi.org/10.7554/eLife.25293.010

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Fractionation, Staining, Sedimentation, Western Blot, Incubation, Quantitative RT-PCR, Control, Expressing, RNA Sequencing, Generated

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Workflow of the experimental strategy used to generate DNase1-digested soluble chromatin. Purified ES cell nuclei were incubated with increasing amounts of DNase1 (10, 20, 40, 70, 100 and 200 units) and for different time points (30 min, 1 hr and 2 hr). DNA was then extracted from the soluble fractions and resolved on an agarose gel. The size distribution of the DNase1-digested chromatin was compared to the nucleosomal ladder obtained after MNase treatment (M.N.). The red arrow indicates the conditions (200 units of DNase1 for 30 min at 15 mM NaCl) that were used to generate DNAse1-solubilized chromatin from the various Suv39h-EGFP rescued ES cell lines ( B ) DNase1-digested and soluble chromatin from Suv39h1-EGFP, Suv39h2-EGFP and Suv39h2-D(T3K81)-EGFP rescued ES cells was either left untreated (upper panels) or incubated with RNaseA at 100 mM NaCl (lower panels) before fractionation on a linear sucrose gradient and Western blot analysis of the sedimentation profile of the various Suv39h-EGFP proteins and endogenous Dnmt3a and HP1α as described for . DOI: http://dx.doi.org/10.7554/eLife.25293.012

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Purification, Incubation, Agarose Gel Electrophoresis, Fractionation, Western Blot, Sedimentation

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: ( A ) Wild type (wt26) and Suv39h double null (dn57) ES cells were separated into cytoplasmic, nucleoplasmic and chromatin fractions and the fractionation profile of Suv39h1, Suv39h2, H3K9me3, histone H3 and Gapdh was analyzed by Western blotting. ( B ) RNA was isolated from the cytoplasmic, nucleoplasmic and chromatin fractions and quantified by RT-qPCR with primers that are specific for major satellite repeats, LINE L1MdA 5'UTR, SINE B1 and SINEB2 repeat elements and for the house-keeping genes Gapdh and Hprt . The relative abundance of each of these transcripts in the three sub-cellular fractions is plotted and is normalized to the total (i.e. the sum of cyt + nuc + chr) RNA signal for each distinct RT-qPCR. Error bars represent the standard deviation from two biological replicates. DOI: http://dx.doi.org/10.7554/eLife.25293.013

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Fractionation, Western Blot, Isolation, Quantitative RT-PCR, Standard Deviation

Journal: eLife

Article Title: Major satellite repeat RNA stabilize heterochromatin retention of Suv39h enzymes by RNA-nucleosome association and RNA:DNA hybrid formation

doi: 10.7554/eLife.25293

Figure Lengend Snippet: Model depicting a higher-order RNA-nucleosome scaffold that is established by chromatin association of major satellite repeat (MSR) RNA. In this model, initial transcriptional activity of the MSR repeats is needed to build heterochromatin. The intrinsic property of MSR repeat sequences to form RNA:DNA hybrids will facilitate their chromatin retention and most likely occurs in inter-nucleosomal regions. Additional portions of ssMSR RNA organize the assembly of a higher-order RNA-nucleosome structure and are also important for the recruitment and stabilization of the Suv39h enzymes to heterochromatin. MSR RNA decorated heterochromatin will provide multiple affinities for the Suv39h KMT, such as ssRNA binding by the basic domain (BD) of Suv39h2 (this study), H3K9me3 and RNA binding by the chromo domains of both mouse Suv39h1 or human SUV39H1 enzymes and HP1 interaction ( ; ). Additional protein-protein contacts with other chromatin-associated components , histone H1 and transcription factors are not shown. DOI: http://dx.doi.org/10.7554/eLife.25293.017

Article Snippet: Recovered proteins from the soluble (supernatant) or insoluble (pellet) fractions were processed for Western blotting with the following antibodies: α-GFP (Invitrogen A11122), α-Suv39h1 (Cell Signalling D11B6) and α-Suv39h2 (LifeSpan BioSciences LS-C116360).

Techniques: Activity Assay, Binding Assay, RNA Binding Assay

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Immunoblot of Yap1 to verify knockout in Yap1 KO cells. β-actin was used as a loading control. ( B ) Representative brightfield microscopy images of WT and Yap1 KO ES cells in ±LIF. Scale bar, 200 μm. ( C ) Immunoblot of Yap1 to verify knockout of Yap1 in three different ESC lines (J1, E14, and CJ7). β-actin was used as a loading control. J1 clone #5 was used as a positive control for knockout. ( D ) RT-qPCR measuring the expression of Yap1 after lentiviral shRNA-mediated Yap1 KD in differentiating WT ESCs (-LIF 72 hr). ( E ) LDH assay measuring cell death of Yap1 KD vs. control KD cells during differentiation (-LIF 72 hr). ( F ) Immunoblot of Yap1 to verify stable overexpression (OE) of FLAG-Bio-Yap1 in three different clones compared to WT ESCs. β-actin was used as a loading control. ( G ) Immunoblot of cleaved Casp3 and cleaved Parp1 in WT and Yap1 KO cells that had been treated with 1 μM STS for the indicated number of hours during differentiation (treatment started 43–48 hr after withdrawal of LIF depending on the length of STS treatment). ( H ) RT-qPCR measuring the expression of Casp9 upon shRNA-mediated lentiviral KD in WT and Yap1 KO cells during differentiation (72 hr) relative to empty vector KD. ( I ) RT-qPCR measuring the expression of Casp2, Casp3, Casp6, Casp7, Casp8, and Casp9 in Yap1 KO cells compared to WT cells in ±LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Western Blot, Knock-Out, Control, Microscopy, Positive Control, Quantitative RT-PCR, Expressing, shRNA, Lactate Dehydrogenase Assay, Over Expression, Clone Assay, Plasmid Preparation, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Lactate dehydrogenase (LDH) assay of WT and Yap1 KO ESCs in ±LIF. Cells were treated with either Z-VAD-FMK (Z-VAD), necrostatin-1, DMSO, or no treatment. Values were normalized to wells that had been lysed completely. ( B ) LDH assay measuring cell death after Yap1 KO in three different ESC lines during differentiation (72 hr) or self-renewal. ( C ) LDH assay measuring cell death in Yap1 KO, WT, and three different stable FLAG-Bio (FB) Yap1 overexpression cell lines during differentiation (72 hr). ( D ) Representative brightfield and fluorescence microscopy images of WT and Yap1 KO ESCs incubated with NucView 488 Casp3 substrate at the indicated times after LIF withdrawal. ( E ) Representative flow cytometry density plots of WT and Yap1 KO ESCs detecting fluorescent signal from annexin-V (conjugated to CF594) and NucView 488 reagent during differentiation (60 hr). ( F ) Fold enrichment of annexin-V and active Casp3-positive Yap1 KO vs. WT ESCs according to flow cytometry. ( G ) Immunoblot of Casp9, Casp8, Casp3, cleaved Casp3, and cleaved Parp1 in WT and Yap1 KO cells during differentiation. β-actin was used as a loading control. ( H ) Luminescent assay of caspase activity in Yap1 KO vs. WT ESCs in ±LIF media. ( I ) LDH assay of WT and Yap1 KO cells ± KD of Casp9 during differentiation (72 hr). All data are expressed as mean ±standard deviation (n = 4 independent samples for LDH assays and n = 3 for other experiments). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Lactate Dehydrogenase Assay, Over Expression, Fluorescence, Microscopy, Incubation, Flow Cytometry, Western Blot, Control, Luminescence Assay, Activity Assay, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Schematic of 3 differentiation protocols (ectoderm, endoderm, and epiblast) used in , and . ( B ) LDH assay of WT and Yap1 KO ESCs in N2B27 with or without 2i and Z-VAD. ( C ) LDH assay of WT and Yap1 KO ESCs in low serum DMEM supplemented with IDE1 ±Z VAD (48 hr). ( D ) LDH assay of ESC towards EpiLC conversion in WT and Yap1 KO ESCs (72 hr). ( E ) Schematic of verteporfin (vert) treatment timings during late and early differentiation in WT ESCs in -LIF. ( F ) Timecourse LDH assay of verteporfin-treated dESCs at the indicated timepoints along with positive controls (treatment with verteporfin just after -LIF as well as untreated Yap1 KO ESCs, the latter of which are n = 8). All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Lactate Dehydrogenase Assay, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Immunoblot of Bcl-2, Bcl-xL, and Mcl-1 in WT and Yap1 KO cells in -LIF after 72 hr of differentiation. ( B ) RT-qPCR measuring the expression of anti-apoptotic (blue) and pro-apoptotic (red) genes in WT ESCs cultured in the indicated differentiation conditions (all at 48 hr) normalized to their respective self-renewal conditions. ( C ) RT-qPCR measuring the expression of anti- and pro-apoptotic genes in Yap1 KO vs. WT cells (log 2 ) in various differentiation conditions (all at 48 hr). ( D ) RT-qPCR measuring the expression of Bcl2 , Bcl2l1 , and Mcl1 in Yap1 KO cells vs. WT cells during differentiation (timecourse). ( E ) RT-qPCR measuring the expression of Bcl2 in WT and Yap1 KO cells during differentiation (timecourse) relative to +LIF. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P. 10.7554/eLife.40167.008 Figure 3—source data 1. Data used in .

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Cell Culture, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Representative confocal images (63X oil objective) of immunocytochemistry of WT and Yap1 KO ESCs in -LIF (72 hr). Blue represents the nucleus, red represents Bcl-2 (top 6) or Mcl-1 (bottom 6), and yellow represents mitochondria. White squares indicate location of zoom images. Scale bar, 20 μm. ( B ) Quantification of fluorescence corresponding to Bcl-2, Mcl-1, and mitochondria using ImageJ, normalized to the number of nuclei in each view as stained by NucBlue. ( C ) RT-qPCR of WT ESCs with transient OE of Yap1 (48 hr) in -LIF (72 hr) normalized to empty vector. Blue indicates anti-apoptotic genes, red indicates pro-apoptotic genes. ( D ) Boxplots of expression of pro-apoptotic genes and anti-apoptotic genes in Yap1 OE cells versus BirA cells (log 2 ) in +LIF. Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. ( E ) Boxplots of expression of pro- and anti-apoptotic genes in Yap1 KD and empty KD in -LIF relative to +LIF (log2). Plus symbols represent the average and middle red bands represent the median. Outliers are represented by hollow circles. Significance stars indicate p-values from paired t-test. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test (unless otherwise specified) compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Immunocytochemistry, Fluorescence, Staining, Quantitative RT-PCR, Plasmid Preparation, Expressing, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Gene feature analysis of Yap1 ChIP-seq quantifying the proportion of Yap1 peaks in promoter, intergenic, upstream, intron, or exon regions. ( B ) Co-IP followed by immunoblot of Tead4 and p300 after pull-down using magnetic streptavidin beads in Yap1 FB cells during differentiation (72 hr). ( C ) Known motif analysis of Yap1 ChIP-seq peaks using BirA ES cells as a background control. Top five motifs with the lowest p-values corresponding to known factors are presented. ( D ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating the presence of the motifs of Tead4, Zic3, and AP-1 complex members JunB and Fra1 (Fosl1). Esrrb is presented as a negative control, as known motif analysis in ( C ) showed no significant enrichment of the Esrrb motif. Input represents 0.03% of total protein lysate. ( E ) Peak-centered histogram of Yap1 ChIP-seq peaks indicating p300 occupancy and H3K27ac presence in ESCs maintained in 2i and EpiLCs, which represent dESCs. ( F ) Gene ontology (GO) analysis of genes bound by Yap1 that are also upregulated (white) or downregulated (black) after Yap1 KD in -LIF (96 hr). ( G ) Schematic of dual luciferase essay using putative Yap1-responsive cis- regulatory elements. ( H ) Schematic of tandem Bcl-2 enhancer creation showing the location of both Yap1-occupied elements that were combined into the same construct. ( I ) Correlation heatmap of YAP1 occupancy on apoptosis-related genes in SF268 glioblastoma cells, NCI-H2052 lung mesothelioma cells, IMR90 lung fibroblasts, and MDA-MB-231 triple negative breast cancer cells. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( J ) Signal tracks of YAP1 occupancy on MCL1 , BCL2 , and BCL2L1 (BCL-XL) in the cell types described in ( I ). ( K ) Correlation heatmap of occupancy of Yap1 in mouse dES cells (from this study), Tead1 in pre-B progenitor cells, Tead2 in Py2T breast cancer cells, and Tead4 in hemogenic epithelium, all on apoptosis-related genes. Genes that were not occupied by any factor were removed from the analysis to reduce noise. ( L ) Signal tracks of Yap1 (red), Tead1, Tead2, and Tead4 (all in blue) on Mcl1 , Bcl2 , and Bcl2l1 (Bcl-xL) in the cell types described in ( I ).

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: ChIP-sequencing, Co-Immunoprecipitation Assay, Western Blot, Control, Negative Control, Luciferase, Construct

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) RNA-seq heatmap (Yap1 KD/empty vector KD, in both undifferentiated and differentiating ESCs) and line graph depicting Yap1 peak score, normalized to BirA, calculated using a moving window average (window = 150). Color bar indicates extent of upregulation (red) or downregulation (green) upon Yap1 KD. ( B ) ChIP-seq peak heatmaps using coordinates centered on the top Yap1 peaks (p-value cutoff, 1e-5) in dESCs (-LIF), which are shown in the second heatmap from the left. The other heatmaps represent occupancy of Yap1 in ESCs (first) or p300 in ESCs (third) or dESCs (fourth) corresponding to Yap1 dESC peak centers ± 3 kb (bin size = 100). ( C ) Signal tracks of Yap1 (red) and p300 (blue) occupancy on apoptosis-related genes in dESCs and EpiLCs, respectively. ( E and F ) Dual luciferase assay of Yap1-occupied cis-regulatory elements from anti- and pro-apoptotic genes in ( E ) Yap1 KO and WT cells ± LIF (48 hr) or ( F ) WT cells with Yap1 or empty OE (in -LIF, 48 hr), relative to pGL3-promoter, 24 hr after transfection. ( G ) Dual luciferase assay of Bcl-2 and Mcl-1 regulatory elements in Yap1 KO cells after transfection of empty vector or vectors containing FLAG-Bio Yap1 with or without a Ser79Ala mutation. ( H ) Dual luciferase assay of Mcl-1 with a deletion of its Tead binding motif (GGAAT on the reverse strand) in WT ESCs ± Yap1 OE. All data are expressed as mean ±standard deviation (n = 3 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P. 10.7554/eLife.40167.011 Figure 4—source data 1. Data used in , .

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: RNA Sequencing, Plasmid Preparation, ChIP-sequencing, Luciferase, Transfection, Mutagenesis, Binding Assay, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) JC-10 mitochondrial membrane potential assay in WT and Yap1 KO cells during various forms of differentiation (72 hr for Pan and EpiLC, 48 hr for Neural and Endo) and self-renewal (maintained for an equal amount of time). Values (525/570 nm ratio, n = 6) corresponding to loss in ∆ψ (mitochondrial membrane potential) in Yap1 KO cells were normalized to WT cells. ( A ) JC-10 assay in WT and Yap1 KO cells in ±LIF after 12 hr of treatment with BH3 mimetics ABT-737, Venetoclax, A-1210477, and A1155463 (total differentiation time: 36 hr). Values (525/570 nm ratio) corresponding to loss in ∆ψ were normalized to DMSO as a control. ( C ) LDH assays of BH3 mimetic dose response curves after 24 hr of treatment in WT and Yap1 KO cells in ±LIF (48 hr differentiation). ( D ) LDH assay of WT and Yap1 KO cells after KD of Bmf or Puma in -LIF conditions (72 hr). ( E ) LDH assay of inducible Bmf and Puma OE (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells in ±LIF (48 hr differentiation). ( F ) Immunoblot of cleaved Casp3, cleaved Parp1, and Mcl-1 in WT and Yap1 KO dESCs (28 hr) after 4 hr of treatment with BH3 mimetics A-1210477 (Mcl-1 inhibitor) and ABT-737 (inhibitor of Bcl-2, Bcl-xL, and Bcl-w). β-actin was used as a loading control. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Membrane, Control, Lactate Dehydrogenase Assay, Western Blot, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) RT-qPCR measuring the expression of Bmf and Puma in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD (n = 3). ( B ) Immunoblot of Bmf and Puma after KD in Yap1 KO cells during differentiation (48 hr) relative to empty vector KD. β-actin was used as a loading control. ( C ) RT-qPCR measuring the expression of Bmf and Puma in WT and Yap1 KO ESCs ± Dox (24 hr) in +LIF (n = 2). ( D ) Immunoblot of Bmf and Puma after OE in WT ESCs in +LIF. β-actin was used as a loading control.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Quantitative RT-PCR, Expressing, Plasmid Preparation, Western Blot, Control

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) Immunoblot of Yap1 and Bcl-xL after OE in Yap1 KO cells relative to WT or empty vector Yap1 KO in +LIF. β-actin was used as a loading control. ( B ) Representative brightfield and fluorescent microscopy images of Yap1 KO cells showing ZsGreen fluorescence ±Dox. Scale bar, 400 μm. ( C ) RT-qPCR measuring the expression of Bcl-2 in WT and Yap1 KO cells during differentiation (72 hr)±Dox (48 hr, 500 ng/mL). ( D ) Immunoblot of Bcl-2 in Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( E ) Immunoblot of Taz in WT and Yap1 KO cells ± Dox (48 hr, 500 ng/mL) in +LIF. ( F ) Immunoblot of Bcl-xL and Mcl-1 after siRNA KD in WT cells in -LIF (48 hr). ( G ) Immunoblot of Bcl-2 after shRNA KD in -LIF (72 hr). ( H ) Quantification of fold increase in cell death from observed upon KD of Bcl-xL, Mcl-1, or Bcl-2, relative to control KD, in -LIF (72 hr). ( I ) RT-qPCR measuring the expression of lineage markers in WT cells transfected with siRNA against Bcl-xL or Mcl-1 in -LIF (72 hr). Expression is indicated as a fold change compared to control siRNA. ( J ) RT-qPCR measuring the induction of lineage markers in WT cells transduced with shRNA against Bcl-2 in -LIF relative to +LIF (72 hr). All data are expressed as mean ±standard deviation (n = 3 independent samples). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Western Blot, Plasmid Preparation, Control, Microscopy, Fluorescence, Quantitative RT-PCR, Expressing, shRNA, Transfection, Transduction, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet: ( A ) LDH assay of WT, Yap1 KO, and Yap1 KO constitutively overexpressing Bcl-xL or Yap1 in -LIF (72 hr). ( B ) LDH assay of inducible Bcl-2 (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells -LIF (72 hr). ( C ) LDH assay of inducible Taz (±Dox, 48 hr, 500 ng/mL) in WT and Yap1 KO cells ± LIF (72 hr differentiation). ( D ) Immunoblot of cleaved Parp1, cleaved Casp3, Bcl-xL, and Mcl-1 in Yap1 KO cells inducibly overexpressing Taz (±Dox, 48 hr, 500 ng/mL) in -LIF (72 hr). ( E ) LDH assay of WT ESCs during differentiation (72 hr) after 48 hr KD of Bcl-xL or Mcl-1. ( F ) LDH assay of WT ESCs ± LIF (72 hr)±KD of Bcl-2. ( G ) RT-qPCR measuring the expression of lineage markers (trophectoderm: Cdx2 and Gata3 , ectoderm: Nes and Otx2 , endoderm: Gata4 , mesoderm: Gsc and T ) in WT and Yap1 KO cells in -LIF (72 hr, n = 3). Expression is indicated as a fold change in +Dox samples relative to -Dox. ( H ) Model proposing roles for Yap1 specific to the exit from self-renewal. In complex with Tead factors like Tead4, Yap1 co-activates anti-apoptotic genes and mildly co-represses pro-apoptotic genes to dampen mitochondrial priming, which thus prevents hyperactivation of the apoptotic cascade through Casp9. All data are expressed as mean ±standard deviation (n = 4 independent samples unless otherwise stated). Two sample two-tailed t-test compared to WT or whatever is specified on the y-axis: *=0.05 > P > 0.01. **=0.01 > P > 0.001. ***=0.001 ≥ P.

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Lactate Dehydrogenase Assay, Western Blot, Quantitative RT-PCR, Expressing, Standard Deviation, Two Tailed Test

Journal: eLife

Article Title: Yap1 safeguards mouse embryonic stem cells from excessive apoptosis during differentiation

doi: 10.7554/eLife.40167

Figure Lengend Snippet:

Article Snippet: Primary antibodies (purchased from Cell Signaling Technology unless otherwise specified) along with dilutions used were the following: β-actin (Abgent #AM1829b, 1:20,000), Yap1 (Santa Cruz Biotechnology #sc-101199, 1:1000), Casp8 (#4927S, 1:1000), Casp3 (#9662S, 1:1000), Cleaved Caspase-3 (#9661S, 1:1000), Cleaved Parp1 (#9548S, 1:1000), Caspase-9 (#9508S, 1:1000), Bcl-2 (#3498S, 1:1000), Bcl-xL (#2764S, 1:1000), Mcl-1 (#94296S, 1:1000), Tead4 (Abcam #ab58310, 1:5000), Puma (Santa Cruz Biotechnology #sc-374223, 1:500), and Bmf (Bioss #bs-7587R, 1:1000).

Techniques: Transfection, Recombinant, Knock-Out, LDH Cytotoxicity Assay, SYBR Green Assay, Membrane, Luciferase, Binding Assay, Software, Microscopy