Fig. 5.

Spatial reorganization of histone modifications during erythroblast differentiation. Immunofluorescence microscopy of FVA cells before (0h) and after (48h) erythropoietin induction stained with Hoechst (blue, panels 1–4, 13, 16) for DNA or Hoechst plus antibodies against histones H3me₂K9 (red, panels 5, 6), H4acK12 (green, panels 7, 8), and H3me₃K9 (red, panels 14, 17) as indicated. Panels 1–4, 13, 16 show original images. Panels 5–8 and 14, 17 show overlayed images obtained after Autodeblur deconvolution (see Materials and Methods). Panels 9–12, 15 and 18 show line profiles (deconvolved images) illustrating the spatial fluorescence intensity changes of the specific antibody staining (red or green channel as indicated) and Hoechst (blue channel) plotted along the paths shown by the yellow lines on panels 5–8, 14 and 17. Scale bar, 10 μm. Panel 19 shows a histogram of average distances between Hoechst peaks and each of the three epigenetic histone modifications: H3me₃K9, H3me₂K9, and H4acK12. Distances were measured on multiple line profiles (such as those shown on panels 9 – 12, 15, 18) recorded after immunofluorescence deconvolution microscopy of FVA cells before (0h) and after (48h) erythropoietin induction. From 40 to 50 measurements were made for each category. Error bars show Standard Deviations. P-values shown over the brackets represent probability associated with a Student’s two-sample unequal variance t-Test with a two-tailed distribution.