Paraformaldehyde (pfa)

Neutrophils in assay media were dispensed into polypropylene tubes (10⁶ cells in 500 μl) and heated in a constant-temperature water bath at 40°C for 40 min or at 43°C for 10 min (heat shock). Some of the samples after heat treatment were then incubated at 37°C for different time intervals. Intracellular levels of HSP70 were then determined by indirect immunofluorescent staining followed by flow cytometry analysis. For intracellular labeling the neutrophils were fixed and permeabilized in DPBS containing 2% paraformaldehyde (Riedel-de Haen, Germany), 0.05% BSA and 0.05% Triton X-100 (Sigma-Aldrich, USA) at 37°C for 15 min. The permeabilized neutrophils were treated in 100 μl volume with primary HSP70-specific monoclonal antibody BRM22 (Sigma-Aldrich, USA) or in HSP70-specific B-hybridoma supernatants in 1:100 dilutions for 30 min at RT and then stained with secondary sheep anti-mouse IgG Fab-fragments conjugated with PE (Sigma-Aldrich, USA) for 30 min at RT. Each stage of labeling was followed by two washes with DPBS containing 0.2% BSA and 0.1% Triton X-100. The cells were finally resuspended in DPBS and analyzed by flow cytometry. HSP70 intracellular levels were determined as means of fluorescence intensity (MFI) corrected for background fluorescence of the negative controls.

Unless otherwise specified, all chemicals and reagents, the ANTI-FLAG M2 affinity gel, the Immobilon-P PVDF Membrane (pore size: 0.45 μm) and Immobilon Western Chemiluminescent HRP Substrate were from Merck/Sigma-Aldrich and Millipore (Darmstadt, Germany). All cell culture media and supplements, the TurboFect transfection reagent, the Amplex^® Red Cholesterol assay kit, the CellLight™ Golgi-RFP, BacMam 2.0 Golgi-labelling reagent, and the eight-well microscopy plates Nunc™ Lab-Tek™ II Chambered Coverglass were from Thermo Fisher Scientific (Waltham, MA, USA). Paraformaldehyde was from Riedel-de Haën (Seelze, Germany), the RC-DC Protein Assay kit from BioRad (Hercules, CA, USA), the ProSieve^® QuadColor™ protein marker from Lonza (Basel, Switzerland) and the PNGase F was from New England Biolabs (Ipswich, MA, USA).

Liver tissue was embedded in OCT (Sakura Finetek) and cryosectioned into 8 μm thick sample using Leica CM 1900 (Leica). The HCC cells were concentrated on the slide glass by Cytospin (Shandon). The samples were fixed with 4% paraformaldehyde (Sigma Aldrich) and permeabilized with 0.1% saponin (Sigma) for 15 minutes and 15 minutes, respectively. After the removal of culture medium, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X100 (Riedel-de-Haën) for 7 minutes and 15 minutes, respectively. Then the samples were blocked with 4% fetal bovine serum (FBS, diluted in PBS), stained by primary antibody solution at 4°C for overnight, secondary antibody at room temperature for an hour, and stained with Hoechst 33342 for 10 minutes before analyzed by fluorescence microscope or high content IN Cell Analyzer (GE Healthcare) and confocal fluorescence microscopy (Leica). The antibodies used in this study and its dilution condition was shown in Supplementary Table S1.