Fig 2: Densitometry performed on western blot.Densitometry data provided semi-quantitative data for protein abundance. The intensity values of all LGCA cell lines (both EpCAMHigh and EpCAMLow) and all HGCA cell lines (both EpCAMHigh and EpCAMLow) were combined and the average intensity calculated, and these are shown for OCT4 (A), SOX2 (C), NANOG (E), KLF4 (G) and c-MYC (I). The intensity values of all EpCAMLow cell lines (both LGCA and HGCA) and all EpCAMHigh cell lines (both LGCA and HGCA) were combined and the average intensity calculated, and these are shown for OCT4 (B), SOX2 (D), NANOG (F), KLF4 (H), c-MYC (J), EpCAM (K) and a-SMA (L). Individual intensity values were normalized against the loading control a-tubulin before being combined and averaged. Error bars show standard deviation.

Fig 3: Detection of Sox2-MB in differentiated mES cells.(A) mES cells stained for SSEA-1 together with the Sox2-MB (blue dots) and the nonspecific-MB (red dots). (B) SSEA-1 stained differentiated mES cells treated with Sox2-MB (blue dots) were compared to SSEA1 stained undifferentiated mES treated Sox2-MB (red dots). (C) Undifferentiated mES cells and mES cells differentiated by exposure to RA were analyzed with RT-PCR. (D) Four quadrants (Q1, Q2, Q3 and Q4) of the differentiated mES cells were selected by comparing the nonspecific-MB fluorescent signal with the Sox2-MB fluorescent signal. (E) The double-positive sorted cell populations (Q2: Sox2-MB+ and SSEA1+) formed significantly more undifferentiated colonies compared to the positive-negative sorted cell populations (Q1: Sox2-MB- and SSEA1+ Q4: Sox2-MB+ and SSEA1- ), and the double-negative sorted cell population (Q3: Sox2-MB- and SSEA1-). (F) Undifferentiated colonies were positively stained for Sox2, Nanog and SSEA1 (Scale bar = 200 µm). Error bars represent the mean ± SEM. Asterisks denotes statistical significance (n = 3 samples ** p<0.01, n = 4 samples*** p<0.001).

Fig 4: Mitotic degradation of OCT4 results in different patterns of accessibility loss.(A) Experimental strategy used to assess the impact of OCT4 depletion at the M-G1 transition. (B–C) Genome browser tracks of RPKM-normalized accessibility profiles across the cell cycle for one locus that decreases (B) at chr11:6894809–6895533 and one that increases (C) at chr9:41247953–4124841 in accessibility upon transient OCT4 depletion in M-G1. (D) log2 fold-change values of accessibility between MD-OCT4 and MD*-OCT4 (control) cells in different cell cycle phases at all accessible OCT4-bound sites, grouped into four clusters by k-means clustering (see Materials and methods). Each line represents one locus. Red line: mean. (E) Frequency of overlap (bar) and enrichment p-values (white digits) of the canonical OCT4::SOX2 motif in the four clusters as well as in unbound regions. The color shows the number of identified OCT4::SOX2 motifs per region. (F) Average RPKM-normalized OCT4 ChIP-seq signal in untreated ZHBTc4 cells 2 kb around loci in the four clusters. Statistics are available in Supplementary file 1. EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase.

Fig 5: Representative in situ hybridization images of lung adenocarcinoma tissue samples demonstrating expression of mRNA transcripts for stemness-associated markers. Expression of OCT4 ((A), brown), NANOG ((B), brown), SOX2 ((C), brown), KLF4 ((D), brown) and c-MYC ((E), brown) can be observed at the mRNA level. Nuclei were counterstained with hematoxylin ((A–E), blue). Original magnification: 1000×; n = 6.