Fig 1: (A) Epifluorescent microscopy image of a cell nucleus with H3K9me3-positive chromocenters being strongly clustered around nucleoli (asterisk), the nucleus DNA is counterstained with DAPI; (B) H3K9me3-positive chromocenters are represented by high-density regions (bright in the next neighbor image presentation) in single-molecule localization microscopy; (C) confocal section of cell nuclei costained for H3K9me3 and CENPA, identifying the PADs, and the nucleus DNA is counterstained with DAPI; (D) confocal maximum projection image costained for H3K9me3 and CENPA with large PAD clusters (encircled) near nucleolus (Nl) and small PADs (arrowed). Scale bars in panels (A)–(D), 10 μm. (E) H3K9me3 chromocenter numbers versus their size in ST control before adding of HRG; the y axis signifies the number of H3K9me3-positive chromocenters in individual cells, and the x axis signifies the DNA content in these cells as measured by DAPI IFI; the boxed information are as follows: the box to the left shows preapoptotic cells, the box in the center shows the main cluster of 2C cells with highly heterogeneous chromocenter numbers, and the box on the right shows the scarce S-G2 cells.
Fig 2: Verification of the MCF-7-HRG model by IF staining of ErbB2 receptors: (A) ST control; (B) cytoplasmic internalization and clustering of the ErbB2-positive foci after treatment with 50 nM HRG for 60 min. These data are indicative of two independent experiments and four technical replicates. Scale bars, 10 μm.
Fig 3: The transcription levels of pericentric HS3 from chr no. 1 and chromosome no. 9, after HRG treatment, were normalized to ST control cells and expressed in averaged fold induction; two independent experiments, four technical replicates; arrow bars represent the standard deviation of all replicates from two experiments, the ST control normalized to one unit. The p-values were adjusted by multiple t-test correction.
Fig 4: HRG treatment induces unfolding of the H3K4me3-positive chromatin. The results of the measurements of the heterogeneity parameter of the H3K4me3-stained, transcriptionally active chromatin are shown in the green channel using ImagePro Plus 4.5 program. After 15 min of HRG treatment, the active chromatin becomes by half less heterogeneous (smoothened) and remains such at 30 min of HRG treatment. The averages of three independent experiments, with standard deviation.
Fig 5: Schematic correlation of data indicating the hypothesis of functional dependency of the chromatin reorganization and gene expression. The commitment of differentiation in MCF-7 cells after 15 min of HRG treatment. Unfolding of PADs under the critical threshold of silencing inducing critical acceleration of the whole-genome transcription is shown. Figure fragment for transcriptome data republished from (16) representing transcription speed change (nrmsf) of 22,277 genes shows the critical point at the 15 min of HRG treatment.
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