Figure 12.

HSF1 might interact with SIRT1 to bring about neuroprotection. A, HEK293T cells were transfected with GFP, SIRT1-Flag, or cotransfected with SIRT1-Flag and HSF1-Myc for 48 h. GFP transfection was used as a negative control. SIRT1-Flag was immunoprecipitated using the Flag antibody. The immunoprecipitate as well as the whole-cell lysate (WCL) were analyzed by Western blotting using Myc and Flag antibodies. SIRT1-Flag interacted with HSF1-Myc. B, Rat CGNs lysates were collected after treatment with HK or LK medium for 8 h. HSF1 antibody was used to pull down endogenous HSF1 and IgG was used as a control. The immunoprecipitates and WCL were analyzed by immunoblotting using a SIRT1 antibody. HSF1 was found to interact with SIRT1 at the endogenous level in CGNs. Unt, Rat CGNs untreated with HK or LK. C, CGNs were transfected with SIRT1-Flag and HSF1-Myc for 8 h and then treated with HK or LK medium for 24 h. Transfected cells were visualized by immunocytochemistry with Myc and Flag antibodies. SIRT1 and HSF1 were found to colocalize with each other in the nucleus of cells transfected with both of the plasmids. D, Appearance of endogenous HSF1 and SIRT1 was analyzed in rat CGNs and HEK293T cells using HSF1 and SIRT1 antibodies. Immunocytochemistry was used to detect endogenous HSF1 (red) and SIRT1 (green). DAPI was used as a nuclear stain. HSF1 and SIRT1 were found to colocalize with each other endogenously in the nucleus. E, Schematic of wild-type SIRT1-Flag and SIRT1 deletion constructs used in our study (adapted from Kim et al., 2008). F, HEK293T cells were transfected with HSF1-Myc and SIRT1-Flag or with HSF1-Myc and 10 deletion constructs of SIRT1, which contains 60–80 sequential amino acid deletions. SIRT1 or the deletion constructs were immunoprecipitated using Flag antibody or IgG antibody. The immunoprecipitates and WCL were analyzed by Western blotting using the Myc and Flag antibodies. HSF1 did not interact with deletion constructs spanning regions 214–541 aa (SIRT1-Δ4-Δ7) and 610–696 aa (SIRT1-Δ9) of SIRT1. SIRT1-Flag and HSF1-Myc lysates pulled down with IgG were used as negative control. G, Rat CGNs were cotransfected with GFP and pLKO.1 or HSF1 shRNA2 or with SIRT1 and shRNA2 in a ratio of 1:2 for 48 h and then treated with HK or LK medium. Viability of neurons was quantified after 24 h using DAPI staining. The survival rates were normalized to GFP- and pLKO.1-cotransfected cultures treated with HK. Data represent the mean ± SD from three independent experiments. **p ≤ 0.01 compared with neurons cotransfected with GFP and pLKO.1 and treated with HK.