Fig 1: Effects of geldanamycin on gene silencing involving miRNAs.(A) Stability of AKT. To verify the efficacy of geldanamycin (GM) in HeLa cells, AKT that is a HSP90 client was examined in cells treated with various concentrations (nM) of geldanamycin by means of Western blotting. Alpha-tubulin was examined as an internal control. (B) Activation of HSF1 by GM. The pGL4-HSE (black bars) and pGL4-minP (gray bars; as a control) reporter plasmids were co-transfected with phRL-TK vector into HeLa cells. After 18 h-incubation, culture medium was changed to fresh medium containing 10 µM GM or DMSO (vehicle), and after 30 min a half of each treated cells were subjected to a mild hyperthermia at 40°C for 12 h. The expression levels of luciferase reporter genes were examined by a dual luciferase assay, and the activity of Photinus (target) luciferase was normalized to that of Renilla (control) luciferase, and further normalized to the data of pGL4-minP given as 1. The data are averages of three independent experiments and error bars represent standard deviations. (C) Gene silencing profiles in the presence of GM. The constructed reporter plasmids for assessment of gene silencing involving miRNAs were transfected into HeLa cells, and the cells were subjected to the same treatments as in B. The assessment of gene silencing was performed as in Fig. 1C. The data are averages of three independent experiments and error bars represent standard deviations. The upper and lower panels indicate the data in the presence and absence of GM, respectively.