Fig 2: Src activity is necessary for the plasma membrane localisation of CD133. (A) Representative images of CD133 localisation in SK-N-DZ cells with 10% FBS, 30% FBS, and 30% FBS plus the Src/Abl inhibitor, dasatinib. Pericentrin is green, CD133 is red, and DAPI (DNA) is blue. Arrows show centrosomes. Arrowheads show CD133 signals at the pericentrosomal region. (B) Immunoblot of CD133, Src, and Src-Y418-p in SK-N-DZ cells with the treatment indicated above each lane. An immunoblot of γ-tubulin served as a loading control. (C) Quantification of SK-N-DZ cells without pericentrosomal localisation of CD133 shown in (A); mean ± standard error of the mean (SEM) from three experiments, *p = 0.0002, *p = 0.0016, respectively. (D) Representative images of CD133 signals localised at the pericentrosomal region in Caco-2 cells treated with dasatinib. 0.1% DMSO-treated cells are used as a control. Pericentrin is green, CD133 is red, and DAPI (DNA) is blue. Arrows show centrosomes. Arrowheads show CD133 signals at the pericentrosomal region. (E) Immunoblot of CD133, Src, and Src-Y418-p in Caco-2 cells treated as indicated above each lane. An immunoblot of γ-tubulin served as a loading control. (F) Quantification of cells with the pericentrosomal localisation of CD133 in (D); mean ± standard error of the mean (SEM) from three experiments, *p < 0.0001. In this experiment, peripheral colony-forming Caco-2 cells were counted. (G) Representative images of CD133 localisation in SK-N-DZ cells transfected with the control vector, Flag-Src (wild type (WT)), and Flag-Src (dominant negative (DN)). Flag is green, CD133 is red, and DAPI (DNA) is blue. Arrowheads show CD133 signals at the pericentrosomal region. (H) Immunoblot of CD133 and Flag in SK-N-DZ cells transfected with the vector indicated above each lane. An immunoblot of γ-tubulin served as a loading control. (I) Quantification of cells without pericentrosomal localisation of CD133 shown in (G); mean ± standard error of the mean (SEM) from three experiments, *p < 0.0001, *p = 0.001, respectively. (J) Immunoblot of Src and CD133 in Huh-7 cells transfected with control or Src siRNA. An immunoblot of γ-tubulin served as a loading control. (K) Quantification of cells with CD133 signals (>128 pixels) at the pericentrosomal region used in the experiment in (J); mean ± standard error of the mean (SEM) from three experiments, *p = 0.0005. (L) Immunoblot of Src and CD133 in Caco-2 cells transfected with control or Src siRNA. An immunoblot of γ-tubulin served as a loading control. (M) Quantification of cells with CD133 signals at the pericentrosomal region used in the experiment in (L); mean ± standard error of the mean (SEM) from three experiments, *p = 0.0006. In this experiment, peripheral colony-forming Caco-2 cells were counted. All scale bars, 10 μm.

Fig 3: DLGAP1-AS2 upregulated CD151 by interacting with E2F1 to regulate the radiation resistance of rectal cancer stem cells.(A–B) Western blot for measuring the protein levels of CD133, MDR1, BCRP1 and γ-H2AX and AKT/mTOR/cyclinD1 signaling. *P<0.05, **P<0.01, ***P<0.001. The error bar represents the mean ± SD. Each experiment was repeated three times.

Fig 4: Validation of CRISPR/Cas9-mediated DAPK1 knockout in HCT116 colorectal cancer cells.a Western Blot of DAPK1 expression status in various colorectal cancer cell lines. Representative images of two independent experiments are shown. GAPDH served as loading control. b Immunofluorescence staining of DAPK1 (green) in parental HCT116 cells and DAPK1 ko clones. Cells were counterstained with phalloidin for F-actin (red) and nuclear Hoechst (blue). Fluorescence microscopy was performed using a x 100 oil immersion objective. Representative images of two independent experiments are shown. Scale bar = 20 µm. c Protein expression of DAPK1 family members DAPK1, DAPK2, DAPK3, DRAK1, DRAK2 and DAPK1 phosphorylation target pMLC in HCT116 wildtype cells and DAPK1 ko clones were detected by Western Blotting using specific primary antibodies. Representative images of two independent experiments are shown. * images were cropped here; all samples were analyzed on the same SDS-PAGE gel; ** GAPDH blot has been used twice see Fig. 5b, proteins have been loaded on the same membrane. d Western Blot analysis of stem cell markers (CD133, CD44) and EMT markers (epithelial marker: E-cad = E-cadherin, mesenchymal marker: Vimentin). Representative images of two independent experiments are shown.*images were cropped here; all samples were analyzed on the same SDS-PAGE gel. e Representative images of endogenous phospho-ERK1/2 (red) levels of immunostained HCT116 cells and DAPK1 ko clones examined by confocal immunofluorescence microscopy (63x; enlarged: cropped and zoomed in). Cells were nuclear counterstained with Hoechst (blue). Immunofluorescence was repeated in two independent experiments and representative images are shown. White arrows: empty nucleus; dashed arrow: nuclear expression of pERK1/2. Scale bar = 50 µm. f pERK1/2 expression analyzed by Western Blot in cytoplasmic (C) and nuclear (N) protein fractions. Representative images of two independent experiments are shown. GAPDH served as loading control in total and cytoplasmatic protein fractions. Lamin A/C was used for nuclear loading control.

Fig 5: The CD133–DNMT1 interaction upregulates p21 and p27. A) Western blot analysis of FLAG‐DNMT1 in CD133+ cells expressing FLAG or FLAG‐DNMT1(Del(155–163)). GAPDH is used as a loading control. B) By Infinium MethylationEPIC BeadChip arrays, the methylation of 680 annotated genes is increased in CD133+ cells expressing DNMT1 (Del(155–163)) compared to control cells (p < 0.001, Δβ ≥ 0.15). Gene ontology results (top five, according to p value) for 680 genes in which methylation is upregulated in CD133+ cells expressing FLAG‐DNMT1(Del(155–163)) are shown. C) qRT‐PCR quantification of the indicated gene mRNA levels in T21286 CD133+ cells expressing FLAG or FLAG‐DNMT1(Del(155–163)). Data is shown as mean ± SD from three independent experiments; ***p < 0.001, **p < 0.01, *p < 0.05, #p > 0.05, Student's t‐test. D‐E) The methylation rate of p21 and p27 promoters in CD133+ cells and matched CD133‐ cells from T21286 (D) and T12752 (E) are analyzed by bisulfite sequencing. Methylation levels are determined by the ratio of converted C nucleotides to total C nucleotides following bisulfite treatment under CpG island. Results are expressed as mean ± SD from three independent experiments; ***p < 0.001, Student's t‐test. F) Western blot analysis of p21 and p27 expression in T21286 CD133+ cells and CD133‐ cells. GAPDH is used as a loading control. G) Western blot analysis of p21 and p27 expression in T21286 CD133+ cells treated with 2% FBS for 7 days. GAPDH is used as a loading control. H) Chromatin immunoprecipitation (ChIP) assay is performed in CD133+ cells and CD133‐ cells using a DNMT1 specific antibody, followed by PCR amplification of p21 and p27 promoter regions between +250 to position −100. Chromatin (defined as input) and GAPDH products immunoprecipitated by DNMT1 Ab are used as positive and negative control.