Figure 1. Heat shock responses and gene silencing mediated by endogenous miRNAs in HeLa cells exposed to a mild hyperthermia.

(A) Western blot analysis. HeLa cells were exposed to a mild hyperthermia (40°C) as well as normal temperature (37°C). After starting incubation at 40°C, cell extracts were prepared at indicated time (h: hours), and examined by Western blotting using antibodies directed against the proteins indicated. Alpha-tubulin was examined as an internal loading control. (B) Early response of HSF1 to heat shock. An acute phosphorylation of HSF1 immediately after heat shock was examined by Western blotting using the same samples as in A. An arrowhead indicates phosphorylated HSF1. (C) Gene silencing profiles involving endogenous miRNAs. The psiCHECK-2 plasmids (Fig. S1) that carried the complementary sequences of target miRNAs (indicated) were transfected into HeLa cells cultured in 96-well culture plates, and the transfection was duplicated. After 6 h-incubation at 37°C, the culture plates, that is transfected cells, were divided into two groups; and a group was further incubated at 37°C (gray bars) for 12 h, and the other group was cultured at 40°C (black bars) for 12 h. After incubation, dual-luciferase assay was carried out. The activity of the Renilla luciferase (target) was normalized to that of the Photinus luciferase, and further normalized to the data obtained from the cells that were transfected with the psiCHECK-2 empty plasmid as a control. The data are averages of four independent experiments and error bars represent standard deviations. Significant difference between the 37°C and 40°C data was analyzed by Student's t-test (two-tailed; * p<0.05). n.s., no significant difference.