Proteinase k

The liver samples were divided into 5 mm sections for subsequent apoptotic analysis, which was carried out with One Step TUNEL Apoptosis Assay Kit (Beyotime, C1088, China) staining. Tissue sections were permeabilized with proteinase K (Beyotime, ST538, China) at 37 °C for 22 min. After washing with phosphate-buffered solution three times, the TUNEL mixed reagents were added to the sections and incubated in dark conditions at 37 °C for 2 h. We used DAPI (Beyotime, C1002, China) to label the nuclei. The TUNEL-positive cells were visualized using a fluorescent microscope (Nikon Eclipse C1, Nikon, Tokyo, Japan) and were defined by a green color with the DAPI label.

Tissue sections were deparaffinized and rehydrated, and antigen retrieval was performed with either citrate buffer or proteinase K (Beyotime Biotechnology, Shanghai, China). After endogenous peroxidase and nonspecific binding blocking, sections were incubated with anti-cleaved-caspase 3, anti-BiP, and anti-BrdU antibodies (ab6326, Abcam, Cambridge, US). After incubation with rabbit HRP-labeled anti-rat antibody or goat HRP-labeled polymer anti-rabbit antibody (ab6734, ab214880, Abcam, Cambridge, US), a color reaction was performed with a liquid DAB+ substrate chromogen system. Then, the sections were counterstained with Mayer’s hematoxylin, dehydrated and mounted. For calculation of the positively stained cells, five fields were randomly selected and images were taken with a microscope (Ti-E, Nikon, Japan). The number of positively stained cells was counted by two independent researchers using ImageJ software (NIH, US).

The ChIP assay was performed as previously described. Briefly, protein A/G beads (Invitrogen) were precleared with binding buffer containing 0.2 mg of salmon sperm DNA per ml for 6 h. The 293.219 cells were transfected with HA-Hes1 or control vector. At 24 h after transfection, cells were crosslinked with formaldehyde at 1% final concentration for 15 min. Glycine was added to quench the formaldehyde with a final concentration of 125 mM. Cells were lysed with SDS lysis buffer containing a protease inhibitor (PMSF). The lysate was sonicated to shear the chromatin to an average length of 750 bp, and 5 μg of indicated antibodies were added to the lysates and incubated at 4°C overnight. The precleared beads were added into each sample at 4°C for 2 h for immunoprecipitation. To extract the DNA fragments, TE buffer with 1% SDS and proteinase K (Beyotime) was added to the washed precipitates. After incubation at 65°C for at least 6 h for reverse crosslinking, the eluted solution was processed using a DNA extraction kit (Bio-Dev). The specific primers used for ChIP DNA amplification are listed in S2 Table.

The ChIP assay was performed as described previously [20 (link)]. Briefly, KG1a-TIWST1 cells were cross-linked with 1% formaldehyde. DNA was sheared to an average size of 200 ~ 500 bp using the sonicator (#Ymnl-1000Y, Immanuel, Nanjing, China). Immunoprecipitation was performed using antibody against TWIST1 (#ab50887, Abcam). Protein A/G agarose was added and rotated for 4 h. The samples were subsequently treated with proteinase K (#ST532, Beyotime) and RNase A (#ST578, Beyotime). Target DNA was extracted using phenol/chloroform and analyzed by PCR with the primers in Table S2.

One Step TUNEL apoptosis assay kit (C1088, Beyotime, Shanghai, China) was applied to detect cell apoptosis in kidney section according to manufacturer's instruction. Briefly, the tissue sections were pretreated with Proteinase K (ST532, Beyotime, Shanghai, China) working solution after dewaxing and rehydration. Then, TUNEL detection solution was added on the slides and incubated with tissues samples for 60 min at 37 °C in a humidified chamber in the dark. Finally, nuclei were stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) (D212, Dojindo, Kumamoto, Japan) and the TUNEL signals were observed with fluorescence microscope (IX81‐FV1000, Olympus, Tokyo, Japan).