(a) The blood stages of P. falciparum characterized by initial Ring, followed by mature trophozoite and segmented schizont stage. The three developmental stages represent the predominant asexual phase of the malaria parasite. (b) Immunofluorescence analysis of H4K31ac (in red) in asexual stages following 12 hr of treatment with DMSO (vehicle) or FR235222 HDACi. Parasite nuclear DNA was stained with Hoechst (blue). Scale bar, 10 μm. (c) Quantification of the intensity of H4K31ac staining in each P. falciparum nucleus following FR235222 stimulation of asexual stages. The results are represented as mean ± standard deviations from four independent experiments; the number of nuclei quantified was at least n = 25. Asterisks indicate statistical significance of H4K31ac difference between FR235222-treated sample and the corresponding control (DMSO) in the ring, trophozoite and schizont stages, as determined by an unpaired two-tailed Student's t-test, ***p<0.0001; n.s., not significant. (d) and (e) Immunofluorescence analysis of H4K31ac in MEF. DNA was stained with DAPI (blue); the bright foci mark pericentromeric heterochromatin. The signal for H4K31ac, along with H3K27ac, H3K4ac or H3K9ac, is enriched in euchromatic regions as shown in the merge. The mark is excluded from the DAPI dense foci that are associated with H3K9me3 and H4K20me3. Scale bar, 10 μm. Data are representative of three independent experiments.