Phosphatase inhibitor cocktail

To determine the levels of activated Hsf1 in the nucleus, nuclear proteins were extracted using a Nuclear Protein Extraction Kit (Solarbio, Beijing, China; R0050) (31 (link)). The gills were collected 2 h after p38 inhibitor administration and homogenized in a cytoplasmic protein extraction reagent containing 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail (AbMole Bioscience, Houston, TX, USA; M7528). The tissue homogenate was shaken for 20 s using a vortex mixer and placed on ice for 5 min. After four rounds of the above treatment, the homogenate was centrifuged at 13,000 × g for 20 min at 4°C. The resultant pellet was washed three times with PBS and resuspended in a nucleoprotein extraction reagent containing 1 mM phenylmethylsulfonyl fluoride and a phosphatase inhibitor cocktail. The resuspension was processed by another four rounds of alternative shaking and ice bath incubation and then centrifuged at 13,000 × g for 20 min at 4°C. The obtained supernatant was used as the nuclear protein sample and was analyzed by western blotting using specific antibodies.

RIPA lysis buffer (Solarbio, China) containing protease inhibitor cocktail (AbMole, USA) and phosphatase inhibitor cocktail (AbMole, USA) was added to 30 mg of tissue, and then the tissue was ground (40 m/s, 20 s, twice) using a homogenizer (MP Biomedicals, USA). The homogenate was incubated at 4 °C for 30 min and then centrifuged at 16,000 g at 4 °C for 10 min. The supernatant was the tissue protein sample. The protein concentration of the tissue protein sample was determined using a BCA Protein Assay Kit (Solarbio, China), and the protein concentration was adjusted to 4 mg/mL by adding loading buffer (NCM Biotech, China). The protein sample was denatured by incubation at 100 °C for 10 min, and WB was performed to detect the expression of proteins in tumor tissue. Information on the antibodies used in WB experiments is shown in Additional file 1: Table S4.

Human osteosarcoma (U2OS), embryonic kidney 293 (HEK293) and monkey Vero (kindly provided by Yu Chen, Wuhan University, China) cells were maintained in Dulbeco’s Modified Eagle Medium (DMEM) (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (PAN, Aidenbach, Germany), and 1% penicillin–streptomycin (BI, Kibbutz Beit-Haemek, Israel). Human myeloid leukemia mononuclear (THP-1) cells (obtained from the American Type Culture Collection) were cultured in RPMI (HyClone, Logan, UT, USA) with 10% FBS and 1% penicillin–streptomycin. All cells were cultured at 37 °C in a 5% CO₂-humidified incubator. Transient transfections of plasmids were performed with JetPRIME (Polyplus, Illkirch-Graffenstaden, France) for U2OS cells, or Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for HEK293 cells according to the manufacturers’ protocols. For chemical treatments, recombinant human IFN-α (purchased from Cyagen, Santa Clara, CA, USA) were dissolved in sterile distilled water or aqueous buffer containing 0.1% BSA and used at a final concentration of 1000 U/mL. The protease inhibitor mixture cocktail and phosphatase inhibitor cocktail were purchased from AbMole (Shanghai, China), and N-ethylmaleimide (NEM) was purchased from Sigma Aldrich, St. Louis, MO, USA.