Ab7817

For immunocytochemistry, cells were fixed with 4% paraformaldehyde for 20 min at 4 °C. After washing with PBS, the cells were treated with 0.3% Triton X-100 in PBS for 10 min and blocked with PBS containing 3% bovine serum albumin (Bovogen, BSAS0.1) for 1 h at 25 °C. The cells were then treated with primary antibodies at the following concentrations: OCT4 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, SC-5279), NANOG (1:500, Abcam, Cambridge, UK, ab80892), EOMES (1:500, Abcam, ab23345), GATA4 (1:200, Abcam, ab84593), tubulin beta III isoform (TUJ1; 1:500, Millipore, Burlington, MA, USA, MAB1637), smooth muscle actin (SMA; 1:500, Abcam, ab7817), SOX17 (1:500, R&D Systems, AF1924), and CDX2 (1:1250, Abcam, ab76541). The following day, the primary antibodies were removed and the cells washed thrice with PBS for 10 min. Finally, the cells were labeled with fluorescence-conjugated secondary antibodies to detect the primary antibodies at the following concentrations: Alexa Fluor 488 (1:500) and Alexa Fluor 568 (1:500). Lastly, they were treated with DAPI or Hoechst in 0.3% Triton X-100 in PBS for 3 min at room temperature and washed.

Cells were fixed at either (1) room temperature for 10 min using 4% paraformaldehyde (PFA) for Figs. 1 and 2 or (2) room temperature for 20 min using 1.6% PFA for Fig. 6 and Extended Data Figs. 5 and 6. Fixed cells were permeabilized and blocked for 1 h using blocking buffer (5% BSA, 0.1% Triton X-100) and incubated overnight at 4 °C with primary antibody: anti-αSMA (1:100, Abcam, ab7817), anti-periostin (1:50, R&D Systems, AF2966), anti-FLAG (1:100, Sigma-Aldrich, B3111), anti-H1.0 (1:100, Abcam, ab134914), anti-vimentin (1:200, Abcam, ab45939) or anti-lamin A/C (1:200, Abcam, ab8984). Appropriate concentration of secondary antibodies was incubated at room temperature for 1 h. Imaging was performed on a fluorescence microscope (Zeiss Axio Vert.A1) or a confocal microscope (Nikon, A1R, ×60). Nuclei were stained using DAPI. Secondary antibody staining alone was used as a negative control.