Figure 3

Tubastatin A induces heat-shock protein expression by activating heat-shock factor 1. 661W cells were treated with 1, 5 and 10 μM of tubastatin A (TST) for 24 h (a), or in (b) with 10 μM TST for 8 h, or with 200 μM H₂O₂ for 6 h or were preincubated with 10 μM TST for 2 h followed by incubation with 200 μM H₂O₂ for 6 h. ac Tub, acetylated tubulin. α-Tub, α-tubulin. Co, untreated control. Quantitative evaluation of immunoblot analysis revealed a significant increase in heat-shock protein (HSP) 70 level after 8 h (c), while HSP25 was significantly enhanced after 24 h (d); n=4. (e) Heat-shock factor 1 (HSF1) activity was investigated using immunoblot analysis of 661W cell extracts that were treated 10 μM TST for 1, 3 and 6 h, or with 5 μM KRIBB11 (KR) for 6.5 h alone, or preincubated with 5 μM KR for 30 min, followed by incubation with 10 μM TST for 1–6 h. (f) Cell viability MTT assay. Cells were treated as indicated. TST (10 μM) for 8 h or KR (5 μM) for 8.5 h did not influence 661W cell number. H₂O₂ (200 μM) for 6 h led to a strong decrease in cell viability, which was enhanced by pre-incubation with TST for 2 h (TST+H₂O₂). Pre-incubation with KR for 30 min followed by incubation with TST for 2 h followed by treatment with H₂O₂ for 6 h (KR+TST+H₂O₂) did not diminish the protective effect of TST. Experiments were carried out three times with similar results. Data represent the mean±S.D. of one representative experiment with eight replicates and are expressed as the percent of the untreated control, which was set at 100%