Epigenetic regulation of defense genes by histone deacetylase1 in human cell line-derived macrophages promotes intracellular survival of Leishmania donovani
Fig 3
Histone deacetylase 1 (HDAC1) expression and enzyme activity in host cells post L. donovani infection.
THP-1 cells (105cells/ml) were infected with L. donovani (20:1, MOI). The cells were harvested at different time points. A: 6 and 24 h post-infection HDAC1 was immunoprecipitated from infected and unifected host cells, and lysates were analysed for HDAC1 enzyme activity by fluorescent assay kit. The unit of fluorometric absorbance used is RFU (Relative Fluorescence Unit) and was calculated, as mentioned in the methods section. B: RNA was extracted from uninfected (UI) and infected cells (IN) at 6 h, 12 h and 24 h post-infection. HDAC1 expression was quantitated by qRT-PCR. Fold change values of HDAC1 mRNA is represented relative to uninfected condition (UI). Basal levels of HDAC1 expression in uninfected cells was used for data normalization and was taken as 1.0. C: Nuclear extracts of uninfected and infected THP-1 cells were harvested at 6, 12 and 24 h post-infection and lysed. Western blotting was performed using HDAC1 specific antibodies to observe HDAC1 protein expression. The symbols (-) and (+) represent uninfected and infected conditions respectively. H3 was used as housekeeping control to confirm equal loading of protein. Molecular weight markers are mentioned at left hand side of the blot and ‘ns’ refers to non-specific protein bands—D: Densitometric analysis of western blot using ImageJ software and normalized to H3 levels. The results represent the mean ± SD (n = 3). ANOVA was used to determine statistical significance. P-value for significance: * (P≤0.01 to 0.05), **** (P≤0.00001).