Fig 2: OCT4 antibody specificity analysis by western blot analysis. (A) Santa Cruz Sc-8626 OCT4 antibody detected exclusively and specifically one band in the positive controls (ES cells and transfected HEK-293 cells, red arrow). With non-transfected cells and TMSCs no signals were obtained. (B) Santa Cruz Sc-9081 OCT4 antibody detected the OCT4-positive controls (red arrow). Additional non-specific bands were present in OCT4-transfected HEK cells as well as in TMSCs and ES cells. (C) Abcam ab19857 OCT4 antibody detected the OCT4-positive control in transfected cells (red arrow). The ES cell positive control signal showed up only very weakly. Additional unspecific bands were detected in OCT4-transfected HEK cells. The upper unspecific band migrating at ∼50 kDa was also present in non-transfected and SOX2-transfected HEK cells as well as in the nuclear fraction of TMSCs. An additional unspecific band (∼110 kDa) was detected in the cytoplasmic fraction of TMSCs.

Fig 3: OCT4 signals obtained in marmoset monkey ESCs with three different polyclonal antibodies. (A) Abcam ab19857 OCT4 antibody directed against the C-terminus recognizing OCT4A and OCT4B (upper panel). Nuclei were stained with DAPI, which is shown as an overlay in the respective lower panels. Scale bar: 100 µm in all panels. (B) Santa Cruz Sc-9081 OCT4 antibody directed against the N-terminus recognizing only OCT4A. (C) Santa Cruz Sc-8626 OCT4 antibody directed against the N-terminus recognizing only OCT4A.

Fig 4: PFKFB4 is required for CD44ICD-mediated up-regulation of glycolysis and stemness. (A) Analysis of glucose uptake in ZR-75-1 and SKBR3 cells. This graph shows that glucose uptake increases in CD44ICD ectopic expression (ICD-SC) ZR-75-1 and SKBR3 cells versus vector controls (MCS-SC), while glucose uptake decreases in PFKFB4 knockdown ICD (ICD-PFKFB4-sh1 and ICD-PFKFB4-sh2) ZR-75-1 and SKBR3 cells versus SC (ICD-SC). (B) Analysis of lactate secretion in ZR-75-1 and SKBR3 cells. (C) Analysis of ROS levels in ZR-75-1 and SKBR3 cells. (D) Western blot analysis of SOX2, OCT4 and NANOG expression in ZR-75-1 and SKBR3 cells. (E) Flow cytometric analysis of ALDH activity in ZR-75-1 and SKBR3 cells. (F) Sphere formation ability of ZR-75-1 cells. One-way ANOVA was used for statistical analysis and data are shown as mean ± SD. Data are representative of at least three independent experiments.

Fig 5: CD44 knockout enhances stem cell-like characteristics of breast cancer cells. (A) Illustration of the sgRNA genomically targeted sequence in mouse Cd44 locus. Constant exons are depicted as green bars, variant exons are depicted as red bars, and introns are depicted as black lines. PAM: protospacer adjacent motif. (B) qPCR analysis of Sox2, Oct4 and Nanog expression in CD44 knockout (CD44KO) EO771 cells versus wild type controls (WT). Establishment of CD44KO EO771 stable cell line was confirmed by western blot (inset). (C) Western blot analysis of SOX2, OCT4, and NANOG expression in CD44KO EO771 cells versus WT. β-actin serves as a loading control. (D) Flow cytometric analysis of side population (SP) in CD44KO EO771 cells versus WT. (E) Sphere formation ability of EO771 cells in CD44KO EO771 cells versus WT. (F) Western blot analysis of CD44s or CD44ICD re-expression in CD44KO EO771 cells. (G) Tumor volume of EO771-derived CD44 cell lines injected into C57BL/6 mice. (H) Tumor weight of EO771-derived CD44 cell lines injected into C57BL/6 mice. (I) Representative images of lung metastasis (left panel). Quantification of total tumor metastasis to the lung based on incidence of dissemination from primary tumors (right panel). For (D-E), Student's t-test was used for statistical analysis and data are shown as mean ± SD. For (G-I), one-way ANOVA was used for statistical analysis and data are shown as mean ± SEM. Data are representative of at least three independent experiments.