Skip to main content
. 2011 Oct 6;7(10):e1002279. doi: 10.1371/journal.pgen.1002279

Figure 5. Knockdown of CAF-1 p150 impairs distribution of H3 variants and leads to the inhibition of heterochromatin formation.

Figure 5

One-cell embryos were microinjected with siRNA targeting CAF-1 p150 (sip150#1 or sip150#2) or with siRNA targeting EGFP (siEGFP) as a control. (A) The morphology of embryos treated with siRNA at 96 h post-fertilization and the percentage of treated embryos that successfully progressed to the blastocyst stage are shown; n indicates the number of embryos examined. Scale bar, 100 µm. (B) The siRNA-treated embryos were microinjected with each Flag-H3 variant mRNA at the two-cell stage. The mRNAs were microinjected into one blastomere of two-cell embryos. Signals for the Flag-H3 variants were detectable in half of the blastomeres. Embryos that developed to the morula stage were immunostained with anti-Flag antibody, and the DNA was stained with DAPI. Scale bar, 20 µm. (C) Quantitative analysis of Flag-H3.3 staining intensity in the siRNA-treated morulae. The numbers of nuclei examined were 69 and 50 for the siEGFP- and sip150-treated embryos, respectively. (D) Fluorescence intensity analysis of DNA and Flag-H3.3 in siRNA-treated morulae. Arrowheads indicate the typical chromatin regions in which DNA is densely stained. Scale bar, 10 µm. (E) Immuno-DNA-FISH analysis for localizing major satellites and Flag-H3.3 in the nucleus of sip150-treated embryos. Arrows indicate the typical major satellites. Scale bar, 10 µm.