P erk

Cells were cultured in a 12-well plate, and FOXA2, RUNX2, COL1A1, t-ERK and p-ERK were detected using a fluorescence microscope (EU5888; Leica, Wetzlar, Germany). Briefly, cells were fixed in 4% paraformaldehyde (Sigma) for 15 min at room temperature, permeabilized and blocked for 30 min in 0.05% Triton X-100 and 5% bovine serum albumin (BSA). Fixed cells were washed three times with PBS and incubated at 4 °C overnight with anti-FOXA2 (1:400; Cell Signalling Technology), RUNX2 (1:1600; Cell Signalling Technology), COL1A1 (1:500; Abcam, Shanghai, China), t-ERK (1:800; Cell Signalling Technology) or p-ERK (1:200; Cell Signalling Technology). Cells were incubated with a fluorescence-conjugated secondary antibody (Beyotime) at room temperature for 2 h and nuclei were stained with 4′,6-diamidino-2-phenylindole (KeyGen Biotech, Nanjing, China) for 4 min. Samples were then observed and photographed under a fluorescence microscope (Leica).

The cells were cultured under induction medium in a 12-well plate. Runx2, COL1A1, p-ERK and p-Smad1/5/9 were detected using fluorescence microscopy (Leica). Briefly, after 10 min of fixation at room temperature in 4% paraformaldehyde, the cells were blocked for 30 min in 0.04% Triton X-100 and 5% bovine serum albumin and then incubated overnight with anti RUNX2 (1:1,600; Cell Signaling Technology, Shanghai, China), anti-COL1A1 (1:500; Abcam, Cambridge, UK), anti-p-ERK (1:100; Cell Signaling Technology), anti-p-Smad1/5/8 (1:800; Cell Signaling Technology). They were then incubated with a fluorescence-conjugated secondary antibody (Beyotime, China) for 60 min. The cell nuclei were stained with DAPI (KeyGen Biotech, Nanjing, China) for 4 min. Cell immunofluorescence was observed using fluorescence microscopy (Leica, Germany).