Epigenetic regulation of defense genes by histone deacetylase1 in human cell line-derived macrophages promotes intracellular survival of Leishmania donovani
Fig 5
Effect of inhibition of HDACI activity by pharmacological inhibitor NaB on defense gene expression and parasite load in L. donovani infected THP-1 cells.
THP-1 cells (105cells/ml) were incubated with L. donovani (20:1 MOI) for 3 h. Subsequently, the infected (IN) cells were incubated with increasing amounts of sodium butyrate (NaB) and were harvested at 6 h. A: Expression of HDAC1 was measured by qRT-PCR and transcription levels were plotted. The significance of the difference in expression levels was measured in infected (IN) and untreated samples (NaB concentration– 0 mM). B: Total HDAC activity in nuclear extracts of infected cells in the presence of increasing amounts of NaB was detected by fluorescent assay kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was calculated as mentioned in the methods section. The significance of the difference in activity was measured in infected (IN) and untreated samples (NaB concentration– 0 mM). C: Dose dependent impact of HDAC1 inhibitor on the parasitaemia count. Infected THP-1 cells (IN) were incubated in increasing concentrations of NaB. After 6 h, cells were stained and amastigotes enumerated visually. D: Expression levels of the host defense genes, post-infection in the presence and absence of NaB (5 mM) were quantified by qRT-PCR. Results represent the mean ± SD (n = 3). The statistical significance was determined using ANOVA, ns (P>0.05),* (P≤0.01 to 0.05), ** (P≤0.01), *** (P≤0.001), *** (P≤0.0001) and **** (P≤0.00001).