Figure 3

Knock-down of HSP90 leads to reduced phosphorylation of STATs, downregulation of Jaks and reduced proliferation in L428 cells. cHL cell line L428 was transfected with 3 μg siRNA against HSP90a& HSP90b or non-targeting scrambled siRNA (On-Targetplus, Dharmacon) by Nucleofection (Amaxa). Due to high expression levels of HSP90 nucleofection was repeated after 48 h. Cells were harvested 48 h after second nucleofection. Tyrosine phosphorylation of STAT3, -5 and -6 as well as protein expression of HSP90 (rat-anti-HSP90 mAB, Stressgen), STAT3, -5, -6, Jak1, -2, -3, Tyk2 and Actin was analysed by immunoblot. (A) Downregulation of HSP90 by siRNA reduced tyrosine phosphorylation of STAT3, -5 and -6 in L428 cells. (B) Downregulation of HSP90 by siRNA led to reduced protein expression of Jak1, -2, -3 and Tyk2 in L428 cells. (C) Reduced cell proliferation was observed by downregulation of HSP90 in L428 cells. cHL cell proliferation was measured by ³H-Thymidin incorporation after 48 h after second nucleofection. ³H-Thymidin was added 16 h – 18 h before harvesting. The data is depicted as percentages (Scrambled = 100%) and error bars mark the standard deviation of three replications.