Ab92516

Tumor cells (5×10⁵ cells per well) were planted into a 24-well plate precoated with gel (PureCol® EZ Gel, Sigma, 1:150) and treated with BI2536 for 24 h. To analyze the translocation of CRT in immunofluorescence, the samples were fixed in 4% paraformaldehyde (PFA) before permeabilization with 0.1% TritonX-100, and then stained with a rabbit anti-mouse anti-CRT antibody (ab92516, Abcam, 1:100) for 30 min. Subsequently, the cells were stained with a secondary antibody FITC goat anti-rabbit IgG (GB22303, Servicebio, 1:200). The cytoskeleton was incubated with phalloidin (C2207S, Beyotime, 1:1000) and cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI, Beyotime). The fluorescence information was acquired by laser confocal microscopy (NIKON Eclipse Ti). For flow cytometry, treated tumor cells were incubated with anti-CRT and secondary FITC goat anti-rabbit IgG as previously described. Then the samples were prepared into 100 μL of PBS suspension and subjected to FACScalibur detection.

Cells or tissues were added with 50 μL radio-immunoprecipitation assay (RIPA) lysis buffer (containing protease inhibitor) (P0013J, Beyotime, Shanghai, China) and protein was gained and quantified by utilization of the bicinchoninic acid (BCA) kit (PC0020, Solarbio). Later, 50 μg of protein was subjected to electrophoresis, and then transferred to nitrocellulose membranes (66485, Pall Corp, East Hills, NY). The membrane was sealed in 5% skimmed milk powder at ambient temperature for 2 h and probed with primary antibodies against KIF3A (ab11259, 1:2000, Abcam), p21 (ab109520, 1:1000, Abcam), p-p21 (ab47300, 1:1000, Abcam), Bax (ab32503, 1:1000, Abcam), Bcl-2 (#4223, 1:1000, Cell Signaling Technology [CST]), Cle-caspase 3 (ab32042, 1:500, Abcam), CD63 (ab134045, 1:1000, Abcam), CD81 (#56039, 1:1000, CST), Hsp70 (#4876, 1:1000, CST), Calreticulin (ab92516, 1:1000, Abcam) and β-actin (#4970, 1:1000, CST) overnight at 4 °C. The next day, the membrane was further incubated with horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG (ab6721, 1:2000, Abcam) at ambient temperature for 1 h. Afterwards, the membrane was visualized by ECL reagent (BM101, Biomiga Inc), and then BioSpectrum 600 imaging system (Ultra-Violet Products, UK) was processed for detection and analysis. β-actin was used as normalizer.

Cardiac tissues were fixed with 10% formalin solution, gradually dehydrated, embedded in paraffin, and then cut into 4-μm-thick sections. Interstitial fibrosis was assessed by Masson’s trichrome staining. The percentage of interstitial fibrosis was calculated using the ImageJ software (Version 1.8, National Institute of Health (NIH), Bethesda, MD, USA). For immunohistochemical staining, a primary anti-CRT antibody (1:200, ab92516; Abcam, Cambridge, UK) was used. At least five randomly selected microscopic fields (magnification, 200 ×) were captured.