Fitc coupled secondary antibodies

Cells were seeded in 24‐well plates over coverslips (1 × 10³ cells/well) and treated as described in Figure legend. Then, cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton X‐100 in PBS, blocked with 3% BSA/PBS and stained with primary antibodies: rabbit anti‐Ki67 (Abcam), mouse anti‐NF‐κB (Santa Cruz Biotechnology), mouse anti‐β‐catenin (Santa Cruz Biotechnology), mouse anti‐NANOG, rabbit anti‐Oct‐4 and mouse anti‐SOX‐2 (Cell Signaling Technology, Danvers, MA, USA). Samples incubated in 3% BSA/PBS only represented negative controls (Supplementary Figure S3). The corresponding FITC‐coupled secondary antibodies (Sigma‐Aldrich) and 1 ng/mL of nuclear dye DAPI (Sigma‐Aldrich) were added during 2 hours. Samples were examined using an epi‐fluorescent microscope (Olympus, Japan).

Immunofluorescence staining was performed as previously reported [27 (link)]. For that purpose, 3 × 10³ cells/well were seeded in 24-well plates over rounded glass coverslips and treated as described in the Results section (Section 3.3, Section 3.4, Section 3.5). Afterwards, cells were fixed in 4% formaldehyde in PBS, permeabilized in 0.1% Triton X-100 in PBS, blocked with 1% BSA/PBS, and incubated at room temperature for 1 h with the following primary antibodies: rabbit anti-Ki67 (Abcam), mouse anti-NANOG, rabbit anti-Oct4, mouse anti-SOX2, rabbit anti-SIRT1, and rabbit anti-FoxO3a (all from Cell Signaling Technology). Subsequently, samples were washed with PBS and incubated with appropriate FITC-coupled secondary antibodies (Sigma-Aldrich) and 1 ng/mL of the nuclear dye DAPI (Sigma-Aldrich) for 2 h. An epifluorescence microscope (Olympus, Tokyo, Japan) was used for the examination of mounted cell samples.