Figure 5

Downregulation of Hsp90 function by treatment with NCT-50. (a,b) H1299 and H460 cells were treated with NCT-50 for 24 h under hypoxic (a) or normoxic (b) conditions. The level of Hsp90 client proteins such as HIF-1α (a), EGFR, IGF1R, Akt, and MEK1/2 (b) was determined by Western blot analysis. (c,d) H1299 and H460 cells were treated with NCT-50 for 24 h. The mRNA expression of HIF-1α target genes (VEGF, TGFB3, and PDGFB) (c) or non-HIF-1α target genes (DDIT3, PPP1R15A, and TRIB3) (d) were determined by real-time PCR analysis. (e,f) HUVECs were treated with CM obtained from NSCLC cells treated with vehicle or NCT-50 (5 or 10 μM). The proliferation (e) and tube formation (f) of HUVECs were determined as described in Methods. (g) H460 cells were treated with NCT-50, ganetespib (Gane) or PU-H71 (PU) for 24 h under hypoxic conditions. The level of HIF-1α expression was determined by Western blot analysis. The bars represent the mean ± SD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 by Student’s t-test compared with vehicle-treated control group.