Figure 4

Effect of treatment with Antp-TPR in the presence of R11-Hsp70 on the expression of Hsp70. (A) BT20 cells were treated with or without Antp-TPR (20 μM), R11-Hsp70 (10 μM), or a combination of these peptides for 6 h or 18 h and examined by western blotting for the expression of Hsp70 and β-actin using corresponding antibodies (left panel). β-Actin was used as the loading control. BT20 cells were transiently transfected with pHsp70Pro-Luc, and activation of the Hsp70 promoter was assessed by promoter assay using a luminometer 18 h after the treatment with or without Antp-TPR, R11-Hsp70 or a combination of these peptides (right graph). (B) Luminescence images (shown in red) of stably transfected BT20 cells with pHsp70Pro-Luc captured by the LV200 system using an exposure of 10s at 0 h, 8 h, and 16 h after treatment with or without Antp-TPR, R11-Hsp70, or a combination of these peptides. Squares in the luminescence images indicate the region of interest (ROI), in which the luminescence intensity was measured for time-lapse analysis at the single-cell level. All scale bars are 100 μm. (C) Time-course analysis of Hsp70 promoter activation on single-cell imaging. Stably transfected BT20 cells with pHsp70Pro-Luc were treated with or without Antp-TPR, R11-Hsp70, or a combination of these peptides, and time-course analysis was performed using the LV200 system as described in the Materials and Methods section. In all experiments, 0.5 μM 17-allylamino-demethoxygeldanamycin (17-AAG) was used as a positive control for the up-regulation of Hsp70 in cancer cells.