Journal: Development (Cambridge, England)

Article Title: Lineage-specific reorganization of nuclear peripheral heterochromatin and H3K9me2 domains

doi: 10.1242/dev.174078

Figure Lengend Snippet: P19 cells are competent to adopt a neural fate or cardiac fate in a model system of cell-fate choice. (A) Scheme for differentiation of P19 cells. (B) Western Blot (WB) of pluripotency marker (Oct4), neurogenic transcription factor (EphA3), cardiogenic transcription factor (Gata4) and housekeeping marker (Gapdh) during differentiation of P19 cells. (C) Representative images of undifferentiated P19, D3 RA- and D3 DMSO-treated cells. (D) Immunofluorescence of pluripotency factor Oct4 in undifferentiated, D3 RA- and D3 DMSO-treated P19 cells. (E) Flow cytometry of undifferentiated, D3 RA- and D3 DMSO-treated P19 cells stained for Oct4 to determine the percentage of Oct4+ cells. Gray line represents negative control; black line represents Oct4-stained sample. (F) Heatmap representation of RNA-seq of P19 cells during differentiation. bio rep, biological replicate. Normalized RNA-seq expression z-score provided. Scale bars: 250 µm in C; 25 µm in D.

Article Snippet: Anti-Oct3/4 antibody conjugated to PE (560186, BD Pharmingen) was prepared in 20 µl of 1× Perm/Wash buffer and the cell pellet was resuspended in antibody solution to incubate for 30 min at 4°C in the dark.

Techniques: Western Blot, Marker, Immunofluorescence, Flow Cytometry, Staining, Negative Control, RNA Sequencing Assay, Expressing

Journal: Development (Cambridge, England)

Article Title: Lineage-specific reorganization of nuclear peripheral heterochromatin and H3K9me2 domains

doi: 10.1242/dev.174078

Figure Lengend Snippet: Lineage-specific master regulators Mef2c and Myocd are specifically repositioned from nuclear peripheral heterochromatin during cell-fate choice. (A) Immunofluorescence of H3K9me2 and LaminB reveals nuclear peripheral heterochromatin in undifferentiated, D3 DMSO- and D3 RA-treated P19 cells. (B) Quantification of H3K9me2 layer thickness. One-way ANOVA test. n=30 measurements per condition. Error bars indicate s.e.m. (C) Representative western blot of H3K9me2 and H3 in nuclear lysates of undifferentiated, D3 DMSO- and D3 RA-treated P19 cells. (D) Quantification of H3K9me2 (normalized by H3) western blot levels. One-way ANOVA test. n =3 biological replicates. (E) High-resolution 3D immuno-FISH reveals that the Mef2c genomic locus is specifically released from the nuclear periphery in D3 RA-treated, but not in undifferentiated or D3 DMSO-treated cells. Representative single confocal z-slice of immuno-FISH of indicated loci in each cell type immunostained with LaminB. (F) Quantification of 3D distance between Mef2c locus and the nuclear periphery. One-way ANOVA test. ***P<0.001. Dotted line represents the thickness of H3K9me2; red line indicates mean; n, number of alleles counted per condition. For the D3 DMSO-treated condition, Oct4– cells were quantified. (G) Percentage of cells with 0, 1 or 2 alleles of Mef2c at the nuclear periphery. Average number of cells is 31 cells per condition. (H) High-resolution 3D immuno-FISH reveals that the Myocd genomic locus is specifically released from the nuclear periphery in D3 DMSO-treated, but not in undifferentiated or D3 RA-treated cells. Representative single confocal z-slice of immuno-FISH of indicated loci in each cell type immunostained with LaminB. (I) Quantification of 3D distance between Myocd locus and the nuclear periphery. One-way ANOVA test. ***P<0.001. Dotted line represents the thickness of H3K9me2; red line indicates mean; n, number of alleles counted per condition. For the D3 DMSO-treated condition, Oct4– cells were quantified. (J) Percentage of cells with 0, 1 or 2 alleles of Myocd at the nuclear periphery. Average number of cells is 36 cells per condition. n.s., not significant. Asterisks (E,H) highlight the position of the genomic locus. Scale bars: 5 µm in A; 5 µm in E,H.

Article Snippet: Anti-Oct3/4 antibody conjugated to PE (560186, BD Pharmingen) was prepared in 20 µl of 1× Perm/Wash buffer and the cell pellet was resuspended in antibody solution to incubate for 30 min at 4°C in the dark.

Techniques: Immunofluorescence, Western Blot