Compound A increases Hsp70 promoter activity dose-dependently and transiently.

<p>(A) L929sA cells, stably transfected with a mHsp70i-luc reporter gene construct, were left untreated (Untr) or were stimulated with heat shock at 43°C for 2h, followed by a recovery period of 2h at 37°C (HS+Rec). Normalized luciferase levels were presented as relative reporter gene activity with the condition ‘Untr’ set as 1. Statistical analysis (unpaired t-test) was performed. This figure is representative for 6 independent experiments. (B) L929sA cells, stably transfected with mHsp70i-luc, were induced for 8h with various concentrations of CpdA, as indicated. All samples were controlled to a similar amount of solvent. This figure is representative for 4 independent experiments. (C) L929sA cells, stably transfected with mHsp70i-luc, were treated with solvent (Solv) or CpdA (10µM) for 6h, 24h or 48h. Data were presented as relative reporter gene activity with the Solv condition set as 1. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to explore if Solv differs from CpdA treatment for each respective induction time. (D) A549 cells, were treated with solvent or CpdA 10µM for the indicated time period. Total cellular mRNA was subjected to RT-qPCR detecting gene expression levels for HSPA1A, normalized using housekeeping 36B4 and β-actin mRNA levels. Four independent experiments with slightly varying time kinetics all show comparable results. (E) PC-3 cells were starved for 48h in 0% DMEM, after which these cells were left untreated or treated with Compound A (10µM), as indicated. Purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin, GAPDH and 36B4. (B)(D)(E) Solv condition was set as 1 and results recalculated accordingly. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to compare with Solv (ns not significant; ** p<0.01; *** p<0.001).