Figure 2

17-AAG inhibits phosphorylation of STAT1, STAT3, STAT5 and STAT6 (A + B), reduces expression of Jak1, Jak3 and Tyk2 (C) and cHL cell proliferation (D). cHL cell lines L428, L1236 and HDLM2 were treated with DMSO (Sigma-Aldrich) or 5 μM 17-AAG (17-(Allylamino)-17-demethoxygeldanamycin, Merck/Calbiochem) for 24 h. STAT tyrosine phosphorylation and Jak expression was analysed by immunoblot as described recently [2]. (A + B) Phosphorylation of STAT3 (Tyr705) (#9131, Cell Signaling Technology), STAT6 (Tyr641) (#9366, Cell Signaling Technology) (A), STAT1 (Tyr701) (#9172, Cell Signaling Technology) and STAT5 (Tyr694) (#9351, Cell Signaling Technology) (B) is completely abrogated within 24 h. STAT1 tyrosine phosphorylation was only detectable in HDLM2 cells. Protein levels of the STAT molcules remain mostly unaltered (Antibodies: STAT1 (#9171) STAT3 (#9132) (Cell Signaling Technology); STAT5 (610191), STAT6 (610291) (BD Transduction Laboratories)) (C) 17-AAG leads to strong reduction of Jak1, Jak3 and Tyk2 expression in L428, L1236 and HDLM2 cells. Jak2 is enhanced in L428 and L1236 cells and reduced in HDLM2 cells. (D) Incubation of cHL cells with 17-AAG leads strong inhibition of cHL cell proliferation. cHL cell proliferation was measured by ³H-Thymidin incorporation after 24 h of 17-AAG treatment. ³H-Thymidin was added 16 h – 18 h before harvesting. The data is depicted as percentages (DMSO = 100%) and error bars mark the standard deviation of three replications.