Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Protein ratios of FLAG-IP (TET1)versus Control-IP (empty vector) AP-MS in two replicates with reciprocal SILAC labeling are plotted, and a few proteins in the TET1 interactome are indicated. (B and F) Co-immunoprecipitation (co-IP) of TET1 partners (B) or TET1/2 (F) by FLAG-IP followed by Western blot analysis in ESCs. (C and D) Co-IP by endogenous PSPC1 (C) and TET1 (D) antibodies followed by western blot analysis in ESCs. (E) Western blot analysis in Tet1/2/3 triple-KO ( Tet TKO) ESCs rescued with FLAG-tagged TET1 or TET2 in ESCs. (G) DNA 5mC and 5hmC dot-blot analysis of WT and Pspc1 KO (two independent clones, C4 and C9) ESCs. dsDNA antibody is reblotted as the loading control. Dnmt1/3a/3b triple-KO ( Dnmt TKO) and Tet TKO ESCs serve as negative controls of 5mC and 5hmC, respectively. (H) UHPLC-MS/MS quantification of 5′-methyl-deoxycytidine (5mC) and 5′-hydroxymethyl-deoxycytidine (5hmC) over deoxycytidine (dC) from genomic DNA of WT and Pspc1 KO ESCs. Experiments were performed in biological duplicates with technical triplicates; p value is from two-tailed t test, and “n.s.” denotes statistically non-significant.

Article Snippet: The membrane was dried, auto-crosslinked with 1200 × 100 μJ/cm 2 , and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the Western blot analysis.

Techniques: Plasmid Preparation, Labeling, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Dot Blot, Clone Assay, Tandem Mass Spectroscopy, Two Tailed Test

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The membrane was dried, auto-crosslinked with 1200 × 100 μJ/cm 2 , and blocked with 5% milk/TBST for 1 h. Next, the membrane was incubated with 5mC (Cell Signaling, 28692) or 5hmC (Active Motif, 39769) antibodies, the same as the Western blot analysis.

Techniques: Recombinant, Affinity Purification, Mass Spectrometry, Methylated DNA Immunoprecipitation Sequencing, Software

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: (A) Protein ratios of FLAG-IP (TET1)versus Control-IP (empty vector) AP-MS in two replicates with reciprocal SILAC labeling are plotted, and a few proteins in the TET1 interactome are indicated. (B and F) Co-immunoprecipitation (co-IP) of TET1 partners (B) or TET1/2 (F) by FLAG-IP followed by Western blot analysis in ESCs. (C and D) Co-IP by endogenous PSPC1 (C) and TET1 (D) antibodies followed by western blot analysis in ESCs. (E) Western blot analysis in Tet1/2/3 triple-KO ( Tet TKO) ESCs rescued with FLAG-tagged TET1 or TET2 in ESCs. (G) DNA 5mC and 5hmC dot-blot analysis of WT and Pspc1 KO (two independent clones, C4 and C9) ESCs. dsDNA antibody is reblotted as the loading control. Dnmt1/3a/3b triple-KO ( Dnmt TKO) and Tet TKO ESCs serve as negative controls of 5mC and 5hmC, respectively. (H) UHPLC-MS/MS quantification of 5′-methyl-deoxycytidine (5mC) and 5′-hydroxymethyl-deoxycytidine (5hmC) over deoxycytidine (dC) from genomic DNA of WT and Pspc1 KO ESCs. Experiments were performed in biological duplicates with technical triplicates; p value is from two-tailed t test, and “n.s.” denotes statistically non-significant.

Article Snippet: DNA 5mC , Cell Signaling , Cat. 28692; RRID: AB_2798962.

Techniques: Plasmid Preparation, Labeling, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Dot Blot, Clone Assay, Tandem Mass Spectroscopy, Two Tailed Test

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA 5mC , Cell Signaling , Cat. 28692; RRID: AB_2798962.

Techniques: Recombinant, Affinity Purification, Mass Spectrometry, Methylated DNA Immunoprecipitation Sequencing, Software

Journal: Cell reports

Article Title: A TET1-PSPC1- Neat1 molecular axis modulates PRC2 functions in controlling stem cell bivalency

doi: 10.1016/j.celrep.2022.110928

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: DNA 5mC , Cell Signaling , Cat. 28692; RRID: AB_2798962.

Techniques: Recombinant, Affinity Purification, Mass Spectrometry, Methylated DNA Immunoprecipitation Sequencing, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial Multiomics Analysis Reveals Only Minor Genetic and Epigenetic Changes in Human Liver Cancer Stem-Like Cells Compared With Other Tumor Parenchymal Cells

doi: 10.3389/fcell.2022.810687

Figure Lengend Snippet: Characterization of LCSL cells in liver cancer patients. (A) Representative transition regions at the junction of LCSL cells and HCC cells or CCA cells (EpCAM immunostaining) in P2 tumor tissues. Scale bar, 50 μm. (B) Dot plot shows the percentages of Ki-67-positive cells in the four cell types of 50 PLC patients with obvious LCSL cells . Each dot represents the mean value of five randomly selected equal fields of IHC images from one patient. NCT, non-CSC tumor cells. Data are presented as the mean ± SD. ***, p < 0.001 by Wilcoxon matched-pairs signed rank test. (C) Representative double immunostaining of EpCAM (red) and 5hmC (dark purple) in one HCC sample with LCSL cells. The green, blue, yellow and red arrowheads indicate PDR, PL, LCSL and HCC cells, respectively. Scale bar, 50 μm. (D) Statistical analysis of 5mC, 5hmC and 5fC staining of liver cancer samples. NCT, non-CSC tumor cells. For 5mC and 5hmC, n = 65; for 5fC, n = 34. Data are presented as violin plots with horizontal bars indicating the mean ± SD. ***, p < 0.001 by Wilcoxon matched-pairs signed-rank test; ns, not significant.

Article Snippet: The primary antibodies used were as follows: anti-5mC (1:5000, Cat#: 28692; Cell Signaling Technology), anti-5hmC (1:10000, Cat#: 39769; Active Motif); and anti-5fC (1:1000, Cat#: 61227; Active Motif).

Techniques: Immunostaining, Double Immunostaining, Staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: Spatial Multiomics Analysis Reveals Only Minor Genetic and Epigenetic Changes in Human Liver Cancer Stem-Like Cells Compared With Other Tumor Parenchymal Cells

doi: 10.3389/fcell.2022.810687

Figure Lengend Snippet: An outline of samples used and experiments performed in this study. FFPE and fresh specimens of primary liver cancer patients were collected and screened for LCSL cells by EpCAM IHC staining and tissue/cell morphology. Spatial mutiomics analysis, including in-situ detection of DNA cytosine modifications (5mC, 5hmC and 5fC), WES and WGBS analysis (for P2, P4 and P7 tissues) by CIL-seq, and spatial transcriptomics (for P7 tissues), was performed on those specimens with obvious LCSL cells.

Article Snippet: The primary antibodies used were as follows: anti-5mC (1:5000, Cat#: 28692; Cell Signaling Technology), anti-5hmC (1:10000, Cat#: 39769; Active Motif); and anti-5fC (1:1000, Cat#: 61227; Active Motif).

Techniques: Immunohistochemistry, In Situ