Immunofluorescence Staining of Cells

Cells were cultured in polymer dishes as described previously^{5 (link)}. Cells were fixed for 20 min using 4% (by weight) formaldehyde solution (Sigma-Aldrich 252,549). Subsequently, cells were permeabilized with 0.5% by volume Tween 20 (Sigma-Aldrich P1379) in PBS for 20 min, and then blocked with 1% by weight bovine serum albumin (BSA, Sigma-Aldrich A7906), 22.5 mg/mL glycine in PBST (0.1% by volume Tween 20 in PBS) for 30 min. Cells were incubated with the primary antibodies diluted in 1% BSA in PBST at 4 °C overnight. After that, cells were incubated with the secondary antibodies in 1% BSA at room temperature for 1 h. Finally, the nuclei were stained with DAPI or Hoechst (Thermofisher Scientific R37606 or R37605) fluorescent dyes. The primary antibody used: Atf2 (abcam ab32019), Beta-Catenin (abcam ab19381), GEF-H1 (abcam ab155785), Lef1 (abcam ab137872), Oct4 (abcam ab19857), RNA pol II (abcam ab5408), Smad4 (CST 46,535).