HeLa and T98G cells were cultured in 100-mm dishes at 37°C overnight and then incubated with fresh culture medium containing 0.5% FBS at 37°C for another 24 h. After treatment with helichrysetin at 37°C for 30 min, cells were collected and washed with PBS, then resuspended in PBS with protease inhibitors. The cell suspension was evenly distributed into PCR tubes and heated at 4, 40, 43, 46, 49 and 52°C for 3 min, and then cooled for another 3 min at room temperature. Subsequently, cells were lysed by rapid freeze-thawing. The lysates were centrifuged at 20,000 × g for 20 min at 4°C. Soluble fractions were transferred to new tubes and samples were prepared for western blot analysis as aforementioned.

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