Hsp70 is required to allow the anti-inflammatory activity of Compound A.

<p>(A) A549 cells were transfected with siControl or siRNA targeting HSPA1A and HSPA1B (siHsp70). Total RNA or total protein extracts were prepared 48h post transfection. Purified mRNA was subjected to RT-qPCR detecting HSPA1A or HSPA1B gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S, as presented in the left panel. For siControl-transfected samples gene expression levels were set at 100%. SiHsp70-transfected results were recalculated accordingly. Statistical analysis (unpaired t-test) was performed to show significant difference between siControl and siHsp70 conditions (*** p<0.001). In the right panel, total cell lysates were subjected to Western blot analysis to detect Hsp70 protein levels. Detection of tubulin served as loading control. (B) In parallel with (A), A549 cells were transfected with siControl or siHsp70. 41h post transfection, cells were pretreated with Solv or CpdA (10µM) for 2h, after which ensued a 6h TNF (2000IU/ml) treatment. Purified mRNA was subjected to RT-qPCR detecting IL8 gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S. The condition Solv (siControl) was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference for selected pair wise comparisons (ns not significant; ** p<0.01; *** p<0.001).