Fig. 4.

Decrease in Hsf1 level in senescence is related to SIRT1 downregulation. (A) HSE-luciferase lentiviral construct was expressed in early passage cells and treated with 5 mM NAM overnight, nutlin-3 for 5 days, or infected with shRNA for SIRT1 or HuR. After heat shock, cells were incubated for 6 h before collection, and luciferase activity was measured. The means and ±SEM indicate three independent experiments. (B) Control vector or lentiviral shRNA for either SIRT1 or HuR was expressed in early passage TIG-1 fibroblasts. Cells were selected with puromycin and treated with 10 μM nutlin-3 for 5 days. Levels of Hsf1 were measured by immunoblotting. (C) Lysates of cells described in Fig. 1(A,B) were immunoblotted for Hsf1. (D) Early passage TIG-1 fibroblasts were treated with nutlin-3 to induce senescence. Cells were collected and Hsf1 mRNA levels were measured by qRT-PCR. (E) Hsf1 mRNA half-life was analyzed as in Fig. 3E. The means and ±SEM were calculated from triplicates of two independent experiments. (F) Early passage cells were infected with retroviral empty vector or shRNA against p53 and then treated with 10 μM nutlin-3 for 5 additional days. Cells lysates were immunoblotted for HuR, SIRT1, and Hsf1. (G) Early passage cells were incubated with 10 μM SB and 10 μM nutlin-3 for 5 days, and levels of HuR, SIRT1, and Hsf1 were measured. (H) 10 μM of SB was added for 24 days to WS fibroblasts, and levels of HuR, SIRT1 and Hsf1 were measured. (I) 10 μM of SB was added for 24 days to WS fibroblasts and heat shocked for 35 min at 43 °C. After 6 h incubation, cells were collected and immunoblotted for Hsp70.