Figure 2.

Inhibition of Xenopus HSP70 promoter activity by ET-743. (A Top) The scheme depicts the Xenopus HSP70 promoter used to obtain stable cell lines (28). (A Middle) Dose response of ET-743 on the 3T3-HSP70 pool; black bars are CAT activities in noninduced cells; gray bars represent CAT activities after heat induction. ET-743 was added to exponentially growing cells 2 h before the 2-h shock at 42°C; cells were then placed for 2 h at 37°C to recover. Values are calculated as fold of activation over basal, non-heat-shocked conditions. (A Bottom) Northern blot analysis of CAT mRNA of 3T3-HSP70 cells extracted from untreated cells kept at 37°C (lane 1) and at 1 h after heat induction either without (lane 2) or with pretreatments for 2 h with the indicated concentrations of ET-743 (lanes 3–5). (B) Same conditions as described for A, except that the 3T3-HSP70Ym pool was used. (C) Dose response of ET-743 from experiments similar to the ones described for A and B, except that three individual clones of HSP70 were used. (D) Comparison between the inhibitory curves of ET-743 on the 3T3-HSP70 pool. Cells were pretreated for 2 h as described for A (2h), pretreated for 1 min and then placed at 42°C (I), or trypsinized before adding ET-743 and then immediately (within 1 min) placed at 42°C (T).