Journal: Nature Structural & Molecular Biology

Article Title: Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening

doi: 10.1038/s41594-022-00790-y

Figure Lengend Snippet: a , YFP-XRCC1 localization to telomeres indicated by FAP-mCer-TRF1 after 10 min dye + light (DL) treatment of RPE FAP-TRF1 cells. b , Percent YFP-XRCC1 positive telomeres per nucleus after no treatment (UT) or 10 min DL in wild-type or OGG1ko RPE FAP-TRF1 cells. Error bars represent the mean ± s.d. from the indicated number n of nuclei analyzed from a representative experiment. Statistical analysis by one-way ANOVA (*** P < 0.001). Immunoblot for FAP-TRF1 and OGG1 in extracts from RPE FAP-TRF1 wild-type and OGG1ko cells. Arrow indicates nonspecific band stained by anti-OGG1. c – e , Cell counts of BJ ( c ), RPE ( d ) or primary BJ ( e ) FAP-TRF1 cells obtained 4 days after recovery from 5 or 20 min dye (D) and light (L) alone, or in combination (DL) as indicated, relative to untreated cells. f , RPE FAP-TRF1 cell cycle analysis 24 h after no treatment or 5 min D, L, DL, 20 J m –2 UVC, or 1 h with 2.5 or 10 mM KBrO 3 determined by flow cytometry. g , RPE FAP-TRF1 colony formation efficiency 7–10 days after indicted treatment. h , i , Percent β-galactosidase-positive BJ FAP-TRF1 cells obtained 4 days after the indicated treatments; 2.5 mM KBrO 3 and 50 μM ETP treatments were for 1 h. In c – i , error bars represent the mean ± s.d. from the number of independent experiments indicated by the black circles. Statistical significance was determined by one-way ANOVA (ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). j , Representative image of 5 min DL-treated BJ FAP-TRF1 β-galactosidase-positive cells. Arrows mark positive cells (turquoise). k , Mitochondrial respiration was examined 24, 48 and 96 h after 5 min D, L or DL. Data are means and error bars are ±95% CI from two independent experiments with seven to eight technical replicates each for BJ and RPE FAP-TRF1 cells.

Article Snippet: Protein concentrations were determined with the BCA assay (Pierce) and 10–30 μg of protein was electrophoresed on 4–12% (or 12% for OGG1ko blot) Bis-Tris gels (Thermo) before transferring to polyvinylidene difluoride membranes (GE Healthcare).

Techniques: Western Blot, Staining, Cell Cycle Assay, Flow Cytometry

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A ENCODE ChIP-seq signal in hESCs showing histone modifications (H3K27ac, H3K4me3, and H3K27me3) and CTCF in STX16-ICR. The location of the CUT&RUN amplicon for panel ( B ) is shown at the bottom. B H3K27ac CUT&RUN-qPCR in STX16-ICR. Black, red, and blue bars represent WT, Δmat, and Δpat STX16-ICR hESCs, respectively. C ENCODE ChIP-seq signals in hESCs showing H3K27ac, H3K4me3, and CTCF in the NESP55 and XLαs regions. The locations of the CUT&RUN amplicons for panels ( D , E ) are shown at the bottom. D , E CUT&RUN-qPCR in hESCs. H3K4me3 in the NESP55 region ( D ) and in the putative XLαs promoter region ( E ) are shown. For B , D , and E , data are shown as mean ± SEM, and each dot represents an independent experiment (three biological replicates). Intergroup comparisons were performed by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: ChIP-sequencing, Amplification

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A (Top) Schematic locations of STX16-ICR with an OCT4 motif in intron 4 and the putative XLαs promoter. (Bottom) A UCSC browser screenshot magnifying the putative XLαs promoter region with FANTOM5 CAGE-seq and ENCODE H1ESCs H3K4me3 ChIP-seq signals. Each colored region corresponds to the region shown in the scheme for reporter constructs in ( B , C ). B , C Luciferase assays in WT hESCs. Forty-eight hours after transfection, firefly luciferase counts were measured and normalized by Renilla luciferase counts. B The effect of STX16-ICR on the activity of the full-length or truncated XLαs promoter (XLp) constructs was tested. From top to bottom, a negative control (promoterless), XLp-only, STX16-ICR with XLp, and STX16-ICR with three different truncated XLp constructs, as indicated by the colored regions. C The role of the OCT4 motif within STX16-ICR was tested. From top to bottom, a negative control (promoterless), XLp-only, WT STX16-ICR with XLp, and the OCT4 motif-deleted STX16-ICR with XLp constructs. For ( B and C ), experiments were repeated three times (biological replicates) with three technical replicates, and representative results are shown. Data are shown as mean ± SEM, and intergroup comparisons were performed by one-way ANOVA with Tukey’s post hoc test.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: ChIP-sequencing, Construct, Luciferase, Transfection, Activity Assay, Negative Control

Journal: Nature Communications

Article Title: A biallelically active embryonic enhancer dictates GNAS imprinting through allele-specific conformations

doi: 10.1038/s41467-025-56608-0

Figure Lengend Snippet: A Schematic representation of XLαs exons and a heterozygous SNP in exon 5 (ex5), rs7121, in which T is maternal (Mat) and C is paternal (Pat). Arrows indicate RT-PCR primer locations. B – D Sanger sequencing of rs7121in XLαs RT-PCR products in hESCs. Results obtained from WT ( B ), STX16-ICR paternally deleted (Δpat STX16-ICR) ( C ), and STX16-ICR maternally deleted (Δmat STX16-ICR) ( D ) hESCs are shown. E – K Differentiation of hESCs into proximal renal tubule (PRT)-like cells. E The differentiation protocol. F , G Tetramethylrhodamine (TMR)-labeled parathyroid hormone (PTH) binding to hESCs ( F ) and differentiated PRT-like cells ( G ). Cells were incubated with TMR-PTH for 30 min, and fluorescence intensity was measured using a flow cytometer. H Quantitative PCR results of NESP55 and XLαs mRNAs in WT hESCs (ES, purple) and PRT-like cells (PRT-like, orange). Each dot represents the result of an independent experiment ( n = 4). Data are shown as mean ± SEM. The comparison was done by one-way ANOVA with Tukey’s post hoc test. I – K Sanger sequencing of rs7121 in XLαs RT-PCR products in differentiated PRT-like cells. Results obtained from WT ( I ), Δpat STX16-ICR ( J ), and Δmat STX16-ICR ( K ) cells are shown. L H3K27ac CUT&RUN qPCR in STX16-ICR using WT hESCs (purple) and WT PRT-like cells (orange). M , N 3C-qPCR detecting the interaction between STX16-ICR and the NESP55 region ( M ) and the XLαs region ( N ). Crosslinking frequencies were expressed as normalized values relative to the mean value of STX16-ICR-NESP55 interaction in hESCs. O – R CUT&RUN qPCR assays in WT hESCs (purple) and WT PRT-like cells (orange). O – Q In the NESP55 region, H3K4me3 ( O ), H3K27me3 ( P ), and CTCF ( Q ) were analyzed. R In the AS2 region, H3K4me3 was analyzed. L – R , each dot represents the result of an independent experiment ( n = 3). Data are shown as mean ± SEM. The comparison was done by two-tailed, unpaired t -tests.

Article Snippet: One μl of H3K27ac antibody (Cell Signaling Technology, 8173), 2 μl of H3K4me3 antibody (Cell Signaling Technology, 9751), 2 μl of H3K27me3 antibody (Cell Signaling Technology, 9733), 2 μl of CTCF antibody (Cell Signaling Technology, 3418), 2 μl of anti-OCT4 antibody (Abcam, ab200834), or 5 μl of normal rabbit IgG (Cell Signaling Technology, #66362) were added to 100 μl of cell suspension in antibody-binding buffer and incubated at 4 °C overnight.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Labeling, Binding Assay, Incubation, Fluorescence, Flow Cytometry, Real-time Polymerase Chain Reaction, Comparison, Two Tailed Test

Journal: eLife

Article Title: C/EBPδ-induced epigenetic changes control the dynamic gene transcription of S100a8 and S100a9

doi: 10.7554/eLife.75594

Figure Lengend Snippet:

Article Snippet: Antibody , Anti-H3K27me 3 (C36B11) (rabbit monoclonal) , Cell Signaling Technology , Cat# 9733, RRID: AB_2616029 , ChIP(3 μg).

Techniques: Subcloning, Transfection, Construct, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Recombinant, Control, Genome Wide, CRISPR, Expressing, Plasmid Preparation, Marker, Sequencing, Cloning, Library Amplification, Mutagenesis, In Vitro, In Vivo, Purification, Protease Inhibitor, Immunoprecipitation, Protein Extraction, Isolation, Software

Journal: Nature

Article Title: Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche.

doi: 10.1038/s41586-020-2166-3

Figure Lengend Snippet: Fig. 4 | Epithelial Ptger4 induces Yap nuclear translocation, mediates RSC mobilization and drives tumour initiation. a, b, Wild-type organoids pretreated with or without 10 μM Ptger4 inhibitor were stimulated with 0.1 μM dmPGE2.Western blots for phosphorylated Yap Ser127 (pYap) and total Yap in cytoplasmic lysates (a), Yap and TBP in nuclear lysates (b). Results are representative of two experiments. c, d, Relative expression of Yap target genes18. c, Wild-type organoids treated with 10 μM Ptger4 inhibitor and 0.1 μM dmPGE2 for 13 h. n = 3 to 4 cultures per condition. One-way ANOVA. d, Yap1ΔIEC and Yap1f/f organoids treated with 0.1 μM dmPGE2 for 13 h. Three cultures per genotype per condition. Two-tailed t-test. e, f, Ptger4f/f (n = 3) and Ptger4ΔIEC (n = 3) mice received 14 Gy of abdominal irradiation. On day 3, the ileum was analysed by haematoxylin and eosin staining (e) and immunostaining for lysozyme (f). Results are representative of three independent experiments. Scale bars, 50 μm. g, Immunostaining for Yap in the small intestine of five-week-

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology Animals and other organisms Human research participants Clinical data Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Phospho-YAP (Ser127), clone D9W2I, Cell Signaling 13008, Ref 5/18 - lot: 5 and Ref 10/17 - lot: 5, dilution 1:1000 (Western blot); YAP, clone D8H1X, Cell Signaling 14074, dilution 1:50 (IHC-P), 1:1000 (Western blot); TBP, clone D5C9H, Cell Signaling 44059, Ref 4/18 - lot: 1, dilution 1:1000 (Western blot); beta-actin, clone C4, Santa Cruz sc-47778, lot D0615, dilution 1:2000 (Western blot); Akt (pan), clone C67E7, Cell Signaling 4691, Ref 1/19 - lot: 20, dilution 1:1000 (Western blot); Vimentin-Alexa Fuor 647 rabbit monoclonal, clone D21H3, Cell Signaling 9856, Ref 8/19 - lot 13, dilution 1:800 (IHC-P), 1:200 (IF); COX2 rabbit polyclonal, Cayman 160126, lot 0482857-1, dilution 1:150 (IHC-P); GFP-Alexa Fuor 488 rabbit polyclonal, Thermo Fisher A-21311, dilution 1:200 (IF); CD45.2- Pacific BlueTM rat monoclonal, clone 104, Biolegend 109820, lot: B249623, dilution 1:200 (flow); Lysozyme-FITC, rabbit polyclonal, DAKO EC 3.2.1.17, dilution 1:100 (IHC-P); Dclk1-Alexa Fuor 647 rabbit monoclonal, clone EPR6085, Abcam ab202755, lot GR229365-2, dilution 1:400 (IHC-P); Chromogranin A rabbit polyclonal, Abcam ab15160, lot ZZG021907A, dilution 1:300 (IHC-P); Olfm4 rabbit monoclonal, clone D6Y5A, Cell Signaling 39141, Ref 12/18 – lot: 1, dilution 1:300 (IHC-P); Ly6a/Sca-1-Alexa Fuor 647 rat monoclonal, clone E13-161.7, Biolegend 122517, lot: B249605, dilution 1:400 (IF); Ly6a/Sca-1-PE-Cy7 rat monoclonal, clone D7, Biolegend 108114, lot: B154904, dilution 1:100 (flow); Cd24 Brilliant Violet 421TM anti-mouse monoclonal, clone M1/69, Biolegend 101825, dilution 1:200 (flow); Laminin A1 rat monoclonal, clone AL-4, R&D MAB4656, dilution 1:50 (IHC-P); 3 nature research | reporting sum m ary O ctober 2018 E-cadherin-FITC mouse monoclonal, clone 36/E-Cadherin, BD 612130, lot 8152975, dilution 1:200 (IHC-P) Validation All antibodies have been validated by the manufacturer for the species and the application to be used for as described in the data sheets provided.

Techniques: Translocation Assay, Western Blot, Expressing, Two Tailed Test, Irradiation, Staining, Immunostaining