Anti cytokeratin 19 antibody

Western blotting was performed as described previously.24 Briefly, total protein was extracted from PDAC cell lines in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Rockford, IL, USA) and nuclear proteins were extracted with the Nuclear Extraction Kit according to the manufacturers’ protocol. Aliquots of total protein (12 μg) were electrophoresed on sodium dodecyl sulfate polyacrylamide gels, 10% Tris‐HCL gels (Bio‐Rad Laboratories, Hercules, CA, USA). The separated proteins were transferred to polyvinylidene difluoride membranes (Bio‐Rad Laboratories) and incubated with primary antibodies for 1 hour. Proteins were detected with anti‐HDAC1 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐SNAIL antibody (1:2000 dilution; Abcam), anti‐ZEB1 antibody (1:1000 dilution; Santa Cruz Biotechnology), anti‐Cytokeratin 19 antibody (1:200 dilution; Santa Cruz Biotechnology), anti‐Histone H3 (1:2000; Cell Signaling Technology, Danvers, MA, USA) and anti–β‐actin antibody (diluted 1:4000; Sigma, Tokyo, Japan). The expression relative to actin expression was depicted as a column and measured with ImageJ software (rsb.info.nih.gov/ij).

Immunocytochemistry (ICC) was performed as described previously.15 Briefly, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X‐100, and blocked with 1% BSA for 10 minutes at room temperature. Subsequently, the cells were stained with the following antibodies: anti‐Cytokeratin 19 antibody (1:50 dilution; Santa Cruz Biotechnology) and anti‐HDAC1 antibodies (1:1000 dilution; Abcam). After counterstaining with Hoechst 33342, Trihydrochloride and Trihydrate (Invitrogen) to visualize the nuclei, the slides were analyzed with a confocal fluorescence microscope (BZ‐9000, KEYENCE, Osaka, Japan).