Journal: bioRxiv

Article Title: Reprogramming progressive cells display low CAG promoter activity

doi: 10.1101/2020.03.03.975664

Figure Lengend Snippet: (A) Schematic representation of the experimental design of reprogramming. (B) Downregulation of CAG:H2B-GFP in reprogramming cells as revealed by fluorescence microscopy. mCherry marks OSKM-expressing cells. Scale bar: 100 μm. (C) FACS analysis of the CAG:H2B-GFP MEFs undergoing reprogramming. OSKMmCherry + cells display reduced GFP signal. (D) Western blot of whole cell lysates from bulk MEFs or subsets isolated from reprogramming cultures based on H2B-GFP intensity. With β-tubulin as loading control, the protein levels relative to MEFs are 0.94 and 1.02 for endogenous H2B, 0.70 and 0.53 for β-actin in H2B-GFP high and H2B-GFP low cells, respectively. (E) FACS analysis of the CAG:GFP MEF cells undergoing reprogramming. A fraction of OSKMmCherry+ cells decreased GFP signal as reprogramming continued.

Article Snippet: Quantification of band intensity was done in Image J. β-actin antibody: Abcam, Ab20272 (1:10,000); β-tubulin antibody: Abcam, Ab6046 (1:5,000); H2B antibody: Cell Signaling, 8135 (1:2,000).

Techniques: Fluorescence, Microscopy, Expressing, Western Blot, Isolation

Journal: bioRxiv

Article Title: Reprogramming progressive cells display low CAG promoter activity

doi: 10.1101/2020.03.03.975664

Figure Lengend Snippet: (A) AP staining of iPS colonies at reprogramming day 12. 15% of the highest or lowest CAG:H2B-GFP+ cells were sorted from the mCherry + cells on day 4 and replated on feeder cells to allow further reprogramming. Reprogramming efficiency is quantified on the right. ***: P < 0.001. (B) Immunostaining of iPS colonies at reprogramming day 12 for Nanog. 15% of the highest or lowest H2B-GFP+ cells were sorted from the mCherry + cells on day 4 and replated on feeder cells to allow further reprogramming. Reprogramming efficiency is quantified on the right. ***: P < 0.001. (C) Real-time PCR analysis of core pluripotent gene expression in MEFs, control iPSCs, ESCs, and iPS colonies derived from H2B-GFP low cells sorted at reprogramming day 4. Expression in MEFs is set to 1. (D) AP staining of iPS colonies at reprogramming day 12. 15% highest or lowest CAG:GFP+ cells were sorted from the OSKMmCherry + population on day 6 and replated on feeder cells to allow further reprogramming. Colonies were scored and quantified on the right. ***: P < 0.001. (E) Schematics of reprogramming timeline using the secondary human fibroblast. (F) AP staining and Nanog immunostaining of colonies at reprogramming day 32. The numbers of AP+ or Nanog+ colonies arising from CAG:GFP-high and CAG:GFP-low cells are shown on the right. **: P < 0.01; ***: P < 0.001.

Article Snippet: Quantification of band intensity was done in Image J. β-actin antibody: Abcam, Ab20272 (1:10,000); β-tubulin antibody: Abcam, Ab6046 (1:5,000); H2B antibody: Cell Signaling, 8135 (1:2,000).

Techniques: Staining, Immunostaining, Real-time Polymerase Chain Reaction, Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Reprogramming progressive cells display low CAG promoter activity

doi: 10.1101/2020.03.03.975664

Figure Lengend Snippet: (A) FACS analysis of CAG:H2B-GFP fibroblasts during reprogramming, which was performed as shown in . Cells were stained with Cell Trace Violet dye at day 4, replated and analyzed at day 6. mCherry + and mCherry– cells indicate OSKM-expressing and non-expressing cells, respectively. (B) mCherry + cells shown in (A) are gated according to the violet dye intensity, and the populations are replotted based on H2B-GFP intensity. Dye low cells display low H2B-GFP intensity. (C) Schematics of the experimental design on the correlation between CAG promoter activity and c-Myc-driven cell cycle acceleration. CAG:H2B-GFP fibroblasts were transduced with inducible c-Myc-2A-mCherry. Cells were labeled with Cell Trace Violet dye and induced for c-Myc expression thereafter. (D) FACS analysis of CAG:H2B-GFP fibroblasts shown in (C), before and after induction for c-Myc expression for 2 days. Note that most of the H2B-GFP low cells are mCherry + . (E) FACS analysis of CAG:H2B-GFP fibroblasts treated as shown in (C), without or with Dox induction for 2 days. Dye low cells (oval gate) correlate with H2B-GFP low cells. (F) FACS plot of fresh LKS cells and GMPs from the bone marrow of CAG:H2B-GFP and CAG:GFP transgenic mice.

Article Snippet: Quantification of band intensity was done in Image J. β-actin antibody: Abcam, Ab20272 (1:10,000); β-tubulin antibody: Abcam, Ab6046 (1:5,000); H2B antibody: Cell Signaling, 8135 (1:2,000).

Techniques: Staining, Expressing, Activity Assay, Transduction, Labeling, Transgenic Assay

Journal: bioRxiv

Article Title: Reprogramming progressive cells display low CAG promoter activity

doi: 10.1101/2020.03.03.975664

Figure Lengend Snippet: (A) FACS plot of H2B-GFP low and high cells at reprogramming day 0, 2, 4 and 6. H2B-GFP low cells decrease in size, as indicated by decreased forward scatter (FSC) during reprogramming. (B) Design of reprogramming experiments after selecting cells based on size. Reprogrammable cells were induced for reprogramming, sorted on FSC and SSC at day 0, 2, 4, and 6, and replated on feeder cells to allow for further reprogramming. (C) Quantification of AP+ and Oct4:GFP+ colonies derived as shown in (B). Reprogramming cells with smaller size enrich for reprogramming progressive cells. *: P < 0.05; ***: P < 0.001; n.s.: non-significant. (D) Confocal images of fibroblasts grown on micropatterned surface, immunostained with MKL1 antibody, or stained with DAPI to reveal the nuclei. Micropatterned surfaces include square, disk and dot shape. (E) Quantification of MKL1 intensity in micropatterned fibroblasts. Small indicates cells patterned with dot shape, big indicates cells patterned with square and disk shape. ****: P < 0.0001. (F) Gene set enrichment analysis (GSEA) of differentially expressed genes between CAG high and CAG low cells at reprogramming day 4 (mouse) and day 7 (human). SRF target genes are enriched in the upregulated DEGs between CAG high and CAG low cells of the same species.

Article Snippet: Quantification of band intensity was done in Image J. β-actin antibody: Abcam, Ab20272 (1:10,000); β-tubulin antibody: Abcam, Ab6046 (1:5,000); H2B antibody: Cell Signaling, 8135 (1:2,000).

Techniques: Derivative Assay, Staining

Journal: Learning & Memory

Article Title: 17β-Estradiol regulates histone alterations associated with memory consolidation and increases Bdnf promoter acetylation in middle-aged female mice

doi: 10.1101/lm.034033.113

Figure Lengend Snippet: Dorsal hippocampal histone H3 acetylation is increased by E 2 . ( A ) Bilateral dorsal hippocampal infusion of 5-µg E 2 significantly increased bulk acetyl H3 levels relative to vehicle 30 min ([***] P < 0.001) and 60 min ([**] P < 0.01) later. In contrast, E 2 had no effect on the acetylation of histones H2A ( B ), H2B ( C ), or H4 ( D ). All proteins were normalized to total histone (each bar represents the mean [±SEM] percent change from vehicle). ( Insets ) Representative Western blots of acetylated and total histone protein.

Article Snippet: Membranes were blocked and incubated with the following anti-rabbit primary antibodies overnight at 4°C: acetyl-H2A (Lys 5), acetyl-H2B (Lys 12) (1:1000, Cell Signaling Technology), acetyl-H3 (pan), acetyl-H4 (Lys 12), HDAC1, HDAC2, or HDAC3 (1:1000, Cell Signaling Technology).

Techniques: Western Blot