Figure 3

Effect of PfHz on HSP70 expression. Peripheral blood mononuclear cells (2.5 × 10⁶) were incubated for 2 h at either 37°C (physiological) or 42°C (heat shock treatment) and stimulated with LPS for 1 h. Cells were then treated with PfHz (10 µg/mL) and incubated for different time points (2 to 48 h). (A) Total RNA was isolated and gene expression analysis by qRT-PCR was performed to quantify the HSP70 (1A + 1B) transcripts, using TaqMan® gene expression assays. β-actin was used as an endogenous control to normalize the data by the ΔΔCT method. Data represent average of individuals (n = 3) with each condition performed in triplicate (error bars represent SEM). * indicates P < 0.05 (one-way ANOVA) relative to control conditions at the given temperature. (B) Cell lysates were prepared after harvesting cells from identical experiments at the stipulated time points, and equal amounts of protein were loaded on 4–20% SDS-PAGE to determine the Hsp70 protein levels by immunoblotting. (C) Densitometry analysis was performed to quantify intensity of production (n = 3); error bars represent SEM. Data shown after normalizing Hsp70 relative to β-actin.