Fig. 2.

Effect of HDAC inhibitors on P. falciparum. a Western blot analysis to check the effect of HDAC inhibitors on histone modifications. Trophozoite stage parasites were cultured for 6 h with DMSO (6 h−), 70 nM of Trichostatin A (TSA) or 75 nM of Apicidin (6 h+). The drug was subsequently washed off, and the cells were grown for another 2 h (6 h/2 h), 4 h (6 h/4 h) or 6 h (6 h/6 h). The antibodies used for each blot are shown on the right. b Similar experiment was repeated with TSA treatment for 6 h, subsequent wash off and growth for 0.5 h (6 h/0.5 h), 2 h (6 h/2 h) and 4 h (6 h/4 h). Total protein was extracted from the respective samples for western blot analysis using antibodies against histone H4 or H4K8ac or Pf HDAC1. c Microarray expression analysis of TSA treated cells. TSA or DMSO treatment for 6 h was done in biological triplicates and subsequent wash-off experiments were done in duplicates. The heat map (left panel) shows average log₂ ratios of replicates (TSA minus DMSO) of the genes differentially expressed after 6 h TSA exposure (Student’s t test, P < 0.05). Right panel represents the parasite stage whose genes are maximally expressed as well as the functionally enriched MPM pathways in each of the clusters. d ChIP-on-chip using H4K8ac antibody to assess differential binding upon TSA treatment. All experiments were done in at least duplicates. The heat map on the left shows average log₂ ratios of replicates (TSA minus DMSO) of the probes differentially acetylated after 6-h TSA exposure (Wilcoxon rank sum test, P < 0.05). Graph on the right shows the percentage of probes that are differentially acetylated upon TSA treatment for 6 h. Inset graph shows the differential occupancy across the gene body. e Venn diagram represents the overlap between differential expression and increased H4K8 occupancy in the ORFs due to TSA. Binomial distribution was used to assign P values for up or downregulated genes overlapping with H4K8ac hyperacetylation. Heat map on the right represents the fold change in H4K8ac occupancy or RNA expression of the hyperacetylated genes. Functional pathways (MPM) significantly enriched in the up- or downregulated gene sets are shown