Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: (A) The weak CD133 staining seen at the luminal border of some normal colonic crypts is considered equivocal and interpreted as negative (x200). In contrast, (B) is a case of colorectal carcinoma with strong CD133 staining of the glandular luminal surface (x200) and (C) a case of poorly differentiated colorectal carcinoma demonstrating CD133 staining as tiny cytoplasmic blobs which represent abortive glands (x200). (D) Colorectal carcinoma with deficient DNA mismatch repair (represented by loss of MLH1). Note the lack of immunoexpression of MLH1 in the colorectal carcinoma nuclei in the presence of immunopositivity in the internal positive controls (stromal cells, lymphocytes and nearby normal enterocytes) (x200)

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Staining

Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: CD133 expression and DNA mismatch repair (MMR) status. The CD133 expression and MMR status as per size, TNM stage and differentiation of the colorectal carcinoma ( n = 80).

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Expressing

Journal: PeerJ

Article Title: DNA mismatch repair and CD133-marked cancer stem cells in colorectal carcinoma

doi: 10.7717/peerj.5530

Figure Lengend Snippet: DNA mismatch repair protein (MMR) status and CD133 expression. CD133 immunoexpression and score being stratified by MMR status in right-sided and left-sided colorectal carcinomas.

Article Snippet: Immunohistochemical staining with a rabbit polyclonal antibody to CD133 (1:200; Abcam ab19898, Cambridge, United Kingdom) and detection by the ultraView universal DAB detection kit (Ventana Medical Systems Inc., Tucson, AZ, USA) was carried out on the Ventana Benchmark XT autostainer (Ventana Medical Systems Inc., Tucson, AZ, USA).

Techniques: Expressing

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Effect of Shogaol on the Expression of Intestinal Stem Cell Markers in Experimentally Induced Colitis in BALB/c Mice

doi: 10.1155/2019/5134156

Figure Lengend Snippet: Cytoplasmic and membranous CD133 expression in epithelial cells of the colonic crypts. (a) Minimal, week expression (sum score 0) in mice of control negative group. (b) Focal, weak expression (sum score 1) in control positive group. (c) Focal, weak-moderate expression (sum score 4) deeply within the colonic crypt in group 3 (DSS exposure & 6-TG treatment). (d) Diffuse, weak expression (sum score 3) in group 4 (DSS exposure & 10 mg/kg b.w. Shogaol treatment). (e) and (f) Diffuse, moderate expression (sum score 6) extends into the transient amplifying and apical regions of the colonic crypts in group 5 (DSS exposure & 20 mg/kg b.w. Shogaol treatment) and group 6 (DSS exposure & 40 mg/kg b.w. Shogaol treatment), respectively.

Article Snippet: Following that, the slides were divided into 2 sets: the first set was incubated overnight at 4°C with rabbit anti-CD133 polyclonal Abs (1 : 100, San Francisco Biorbyt, USA), and the second set was incubated with rabbit anti-CD34 monoclonal Abs (1 : 100, Dako, Germany).

Techniques: Expressing, Control

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: Effect of Shogaol on the Expression of Intestinal Stem Cell Markers in Experimentally Induced Colitis in BALB/c Mice

doi: 10.1155/2019/5134156

Figure Lengend Snippet: Immunofluorescence expression of the CD133 protein in the cytoplasm of the colonic cryptal epithelium. (a) and (b) Weak (+) expression in mice of the control negative and control positive group. (c) and (d) Weak-moderate (+ to ++) expression in the in deep portions of the colonic crypts in mice of group 3 (6-TG treatment) and group 4 (10 mg/kg b.w. Shogaol treatment). (e) and (f) Moderate (++) expression in mice of group 5 (20 mg/kg b.w. Shogaol treatment) and mice of group 6 (40 mg/kg b.w. Shogaol treatment).

Article Snippet: Following that, the slides were divided into 2 sets: the first set was incubated overnight at 4°C with rabbit anti-CD133 polyclonal Abs (1 : 100, San Francisco Biorbyt, USA), and the second set was incubated with rabbit anti-CD34 monoclonal Abs (1 : 100, Dako, Germany).

Techniques: Immunofluorescence, Expressing, Control

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Botanical from Piper capense Fruit Can Help to Combat the Melanoma as Demonstrated by In Vitro and In Vivo Studies

doi: 10.1155/2021/8810368

Figure Lengend Snippet: PCFE treatment modulates the expression of EMT markers in vitro . PCFE alone and in combination with dacarbazine upregulates the expression of FITC-tagged (green) E-cadherin and downregulates PE-tagged (red) vimentin of B16-F10 cells as compared to the untreated control (a). Western blot analysis and densitometric plot also showed significant upregulation of E-cadherin and downregulation of vimentin as well as CD133 (b, c). Statistical significance of each treatment group with untreated control is analyzed by one-way ANOVA test ( p ANOVA < 0.0001) followed by post hoc Tukey's test. Data are represented as mean ± SD of triplicate determinations from their independent experiments; ns: not significant, ∗ p value <0.05, ∗∗ p value <0.01, and ∗∗∗ p value <0.0001 versus untreated control and among each group.

Article Snippet: Antibodies vimentin (mouse IgG1; NBP2-32910) and CD133 (rabbit IgG; NB120-16518) were purchased from Novus Biologicals (10730 E. Briarwood Avenue Centennial, CO 80112); E-cadherin (rabbit IgG; sc-7870), β -actin (rabbit IgG; sc-47778), and CD31 (mouse IgG) (sc-1506) were purchased from Santa Cruz Biotechnology (Bergheimer Str.

Techniques: Expressing, In Vitro, Control, Western Blot

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Botanical from Piper capense Fruit Can Help to Combat the Melanoma as Demonstrated by In Vitro and In Vivo Studies

doi: 10.1155/2021/8810368

Figure Lengend Snippet: Qualitative and quantitative effect of PCFE on the expression of the markers that play a key role in the VM and EMT. Immunofluorescence study with various individual treatments of PCFE and in combination with dacarbazine in mice melanoma tumor sample showed an increase in the expression of E-cadherin and decrease in vimentin (a). Western blot analysis (b) and its densitometry plot (c, d) also showed significant upregulation of E-cadherin and downregulation of vimentin and CD133 in tumor tissue lysate of mice melanoma. Statistical significance of each treatment group with untreated control is analyzed by one-way ANOVA test ( p ANOVA < 0.0001) followed by post hoc Tukey's test. Data are represented as mean ± SD of triplicate determinations from their independent experiments with ∗ p value <0.05, ∗∗ p value <0.01, and ∗∗∗ p value <0.0001versus untreated control and among each group.

Article Snippet: Antibodies vimentin (mouse IgG1; NBP2-32910) and CD133 (rabbit IgG; NB120-16518) were purchased from Novus Biologicals (10730 E. Briarwood Avenue Centennial, CO 80112); E-cadherin (rabbit IgG; sc-7870), β -actin (rabbit IgG; sc-47778), and CD31 (mouse IgG) (sc-1506) were purchased from Santa Cruz Biotechnology (Bergheimer Str.

Techniques: Expressing, Immunofluorescence, Western Blot, Control

Journal: PeerJ

Article Title: YAP1 affects the prognosis through the regulation of stemness in endometrial cancer

doi: 10.7717/peerj.15891

Figure Lengend Snippet: HEC1-A, Ishikawa, and RL952 were used for culture of ECSCs. The most stem-like state cell lines were HEC1-A and Ishikawa. These two cell lines were used for the subsequent experiments. Student’s t -test was used to test the differences between two groups. (A) The spheric formation images of HEC1-A, Ishikawa, and RL952 on the 3rd, 5th and 7th day were displayed (100X). (B–C) Analysis of the relative mRNA expressions of SOX2, OCT4, NANOG, CD44, and CD133 in different ECSCs, compared with its original cell lines. (qRT-PCR were tested for three times. Mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.) (D–E) Protein expressions of SOX2, OCT4, NANOG, CD44, and CD133 by western blot in different ECSCs and controls. (F) Relative mRNA expression of YAP1 detected by qRT-PCR. (qRT-PCR were tested for three times. Mean ± SD. *** P < 0.001) (G) Protein expression of YAP1 in different ECSCs, compared with its original cell lines.

Article Snippet: The primary antibodies were as follows: YAP (D8H1X) XP ® Rabbit mAb (1:1000, 14074S, Cell signaling technology), SOX2 Polyclonal antibody (1:1000, 11064-1-AP; Proteintech), OCT4 Polyclonal antibody (1:1000, 11263-1-AP; Proteintech), NANOG Polyclonal antibody (1:1000, 14295-1-AP; Proteintech), CD44 Polyclonal antibody (1:2000, 15675-1-AP; Proteintech), CD133 Polyclonal antibody (1:2000, 18470-1-AP; Proteintech), GAPDH Monoclonal antibody (1:50000, 60004-1-lg; Proteintech), Alpha Tubulin Polyclonal antibody (1:2000, 11224-1-AP; Proteintech).

Techniques: Quantitative RT-PCR, Western Blot, Expressing

Journal: PeerJ

Article Title: YAP1 affects the prognosis through the regulation of stemness in endometrial cancer

doi: 10.7717/peerj.15891

Figure Lengend Snippet: The efficiency of spheres formation was compared after YAP1 knockdown. (A–B) Down-regulation of YAP1 by siRNA was confirmed by qRT-PCR. (qRT-PCR were tested for three times) (C) Images of spheres formation of ECSCs Ishikawa after YAP1 knockdown on the 7th day (100X). (D) Counting of ECSCs Ishikawa spheres. Mean number of five random scopes stood for the ultimate result. (E) Images of spheres formation of ECSCs HEC1-A after YAP1 knockdown on the 7th day (100X). (F) Counting of ECSCs HEC1-A spheres. Mean number of five random scopes stood for the ultimate result. After YAP1 knockdown in Ishikawa and HEC1-A, the expressions of SOX2, OCT4, NANOG, CD44, and CD133 were detected by qRT-PCR and western blot. (G) mRNA expressions of SOX2, OCT4, NANOG, CD44, and CD133 in Ishikawa were detected by qRT-PCR. (qRT-PCR were tested for three times. Student’s t -test was used to test the differences between two groups. Mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.) (H) Protein expressions of YAP1, SOX2, OCT4, NANOG, CD44, and CD133 in Ishikawa were detected. (I) mRNA expressions of SOX2, OCT4, NANOG, CD44, and CD133 in HEC1-A were detected by qRT-PCR. (qRT-PCR were tested for three times. Student’s t -test was used to test the differences between two groups. Mean ± SD. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.) (J) Protein expressions of YAP1, SOX2, OCT4, NANOG, CD44, and CD133 in HEC1-A were detected.

Article Snippet: The primary antibodies were as follows: YAP (D8H1X) XP ® Rabbit mAb (1:1000, 14074S, Cell signaling technology), SOX2 Polyclonal antibody (1:1000, 11064-1-AP; Proteintech), OCT4 Polyclonal antibody (1:1000, 11263-1-AP; Proteintech), NANOG Polyclonal antibody (1:1000, 14295-1-AP; Proteintech), CD44 Polyclonal antibody (1:2000, 15675-1-AP; Proteintech), CD133 Polyclonal antibody (1:2000, 18470-1-AP; Proteintech), GAPDH Monoclonal antibody (1:50000, 60004-1-lg; Proteintech), Alpha Tubulin Polyclonal antibody (1:2000, 11224-1-AP; Proteintech).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot

Journal: PloS one

Article Title: Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a key role in vasculogenic mimicry formation, neovascularization and tumor initiation by Glioma stem-like cells.

doi: 10.1371/journal.pone.0057188

Figure Lengend Snippet: Figure 2. Preferential expression of VEGFR-2 by GSLCs isolated from U87 GBM cells. (A) The expression of mRNAs for VEGFR1, VEGFR-2, VE-cadherin, EphA2, and laminin 5c2 in GSLCs and U87 GBM cells was measured by RT-PCR or real-time RT-PCR. * Indicates significantly increased expression by GSLCs. (B) Western blot of VEGFR-2 (230 and 200 KDa) in GSLCs and U87 GBM cells. b-actin was used as an internal control. (C, D) The effect of VEGF on the expression of mRNAs for VEGFR-2 (C) and VE-cadherin (D) in GSLCs or U87 GBM cells was measured by real-time RT-PCR. * Indicates significantly increased expression of genes compared to U87 cells or by VEGF treated cells (* P,0.01). (E) HE staining of human glioma specimens. IV: WHO Grade IV; III: WHO Grade III; II: WHO Grade II. Co-expression of CD133 (red) and VEGFR-2 (green) by human Grade IV GBM, Grade III anaplastic astrocytoma and Grade II astrocytoma sections is shown. Scale bar = 50 mm. doi:10.1371/journal.pone.0057188.g002

Article Snippet: Primary antibodies used for staining frozen sections were antihuman CD133 (1:30 dilution. sc-30220, rabbit polyclonal, Santa Cruz Biotechnology, USA), anti-human VEGFR-2 (1:100 dilution.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: PloS one

Article Title: Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a key role in vasculogenic mimicry formation, neovascularization and tumor initiation by Glioma stem-like cells.

doi: 10.1371/journal.pone.0057188

Figure Lengend Snippet: Figure 5. The effect of VEGFR-2 shRNA on the self-renewal of GSLCs and their formation of tubules. (A) Formation of spheres by GSLCs with VEGFR-2 shRNA. * Indicates significantly reduced sphere formation by U87 cells containing VEGFR-2 shRNA (p,0.05). (B) VEGFR-2 knockdown by shRNA in GSLCs. RT-PCR analysis of VEGFR-2 mRNA (top) and Western blot of VEGFR-2 (bottom). (C) IF images of spheres formed by VEGFR-2 knockdown GSLCs (upper) or by differentiated VEGFR-2 knockdown GSLCs (lower). IF staining of CD133 (red) and Oct4 (yellow) on spheres is shown in the upper panels. IF staining of GFAP (green) and MAP-2 (red) on differentiated cells is shown in lower panels. (D) RT-PCR of mRNA for VE-cadherin in GSLCs with VEGFR-2 shRNA. * Indicates significantly reduced mRNA in VEGFR-2 knockdown GSLCs compared to Mock shRNA cells (p,0.05). (E) Tubule formation on Matrigel by GSLCs with VEGFR-2 shRNA in the presence or absence of VEGF. Spheres are indicated by white arrows; tubules are indicated by red arrows. Images were taken under light microscopy (6200). (F) Quantitative analysis of tubule formation by VEGFR-2 knockdown GSLCs. * Indicates significantly increased tubule formation by GSLCs containing Mock shRNA in response to VEGF (10 ng/ml) (* p,0.05). # Indicates significantly reduced tubule formation by GSLCs containing VEGFR-2 shRNA. doi:10.1371/journal.pone.0057188.g005

Article Snippet: Primary antibodies used for staining frozen sections were antihuman CD133 (1:30 dilution. sc-30220, rabbit polyclonal, Santa Cruz Biotechnology, USA), anti-human VEGFR-2 (1:100 dilution.

Techniques: shRNA, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Light Microscopy

Journal: PloS one

Article Title: Vascular endothelial growth factor receptor 2 (VEGFR-2) plays a key role in vasculogenic mimicry formation, neovascularization and tumor initiation by Glioma stem-like cells.

doi: 10.1371/journal.pone.0057188

Figure Lengend Snippet: Figure 6. The effect of VEGFR-2 shRNA on tumorigenesis, angiogenesis, self-renewal and VM formation by GSLCs. (A) The growth of xenograft tumors initiated by GSLCs with or without VEGFR-2 shRNA. * Indicates significantly reduced growth of tumors formed by GSLCs with VEGFR-2 shRNA (p,0.05). (B) Survival of mice with xenograft tumors formed by GSLCs with or without VEGFR-2 shRNA. * Indicates significantly prolonged survival of mice bearing tumors formed by VEGFR-2 containing GSLCs (p,0.05). (C) IF images of murine or human CD31 (red) in the xenograft tumors formed by GSLCs with or without VEGFR-2 shRNA. Nuclei were stained with DAPI (blue). Scale bar = 50 mm. (D) Self-renewal of GSLCs with VEGFR-2 shRNA. IF staining of CD133 (red) in the xenograft tumors derived from GSLCs with or without VEGFR-2 shRNA. Nuclei were stained with DAPI (blue). Scale bar = 50 mm. * Indicates significantly decreased number of CD133-positive cells in mice bearing tumors formed by VEGFR-2 knock-down GSLCs (p,0.05). (E) VM formation by GSLCs with or without VEGFR-2 shRNA. IF staining of human LamininB2 (red) or human GFAP (green) in the xenograft tumors derived from GSLCs with or without VEGFR-2 shRNA. Nuclei were counterstained with DAPI (blue). Scale bar = 20 mm. Quantitative image analysis of laminin VM immunoreactivity for glioma derived from Mock or VEGFR-2 shRNA-transfected GSLCs xenografts (n = 6 recipient mice per experimental group). Y-axis, percentage of area with reactivity (mean 6SE, * P,0.01). doi:10.1371/journal.pone.0057188.g006

Article Snippet: Primary antibodies used for staining frozen sections were antihuman CD133 (1:30 dilution. sc-30220, rabbit polyclonal, Santa Cruz Biotechnology, USA), anti-human VEGFR-2 (1:100 dilution.

Techniques: shRNA, Staining, Derivative Assay, Knockdown, Transfection

Journal: International journal of molecular medicine

Article Title: RACK1 affects glioma cell growth and differentiation through the CNTN2-mediated RTK/Ras/MAPK pathway.

doi: 10.3892/ijmm.2015.2421

Figure Lengend Snippet: Figure 7. Relative protein levels of cell differentiation markers glial fibrillary acidic protein (GFAP) and CD133 in cells following transfection with receptor for activated C kinase 1 (RACK1)-pcDNA3.1 and contactin-2 (CNTN2) siRNA. β-actin was used as an internal control. Lane 1, Vec + Scr si; lane 2, Vec + CNTN2 siRNA (si); lane 3, RACK1 + Scr si; lane 4, RACK1 + CNTN2 si. *P<0.05 and #P<0.01 compared with the Vec + Scr si group; &P<0.05 and $P<0.01 compared with the RACK1 + Scr si group. Vec, pcDNA3.1 vector; Scr si, scrambled siRNA.

Article Snippet: After blocking with 5% bovine serum albumin (Amresco LLC, Solon, OH, USA), the membranes were washed with TBST 3 times, and incubated with primary antibodies, namely rabbit polyclonal antibody to RACK1 (1:800; ab62735), rabbit polyclonal antibody to CNTN2 (1:400; ab68994), rabbit polyclonal antibody to epidermal growth factor receptor (EGFR; 1:800; ab2430), rabbit polyclonal antibody to platelet-derived growth factor receptor, α polypeptide (PDGFRα; 1:500; ab65258), and mouse monoclonal antibody to β-actin (1:2,000; ab6276) (all from Abcam, Cambridge, MA, USA), mouse monoclonal antibody to Ras (1:500; sc-166691), rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP) (1:400; sc-9065) (all from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), rabbit polyclonal antibody to CD133 (1:400; EAP0023; Elabscience, Hubei, China), rabbit monoclonal antibody to ERK1/2 (1:400; #4695), rabbit monoclonal antibody to p-ERK1/2 (Thr202/Tyr204; 1:400; #4377) (Cell Signaling Technology, Inc., Beverly, MA, USA) at 4 ̊C overnight, followed by incubation with HRP-conjugated secondary antibodies, namely goat anti-rabbit IgG HRP (1:2,000; ab6721) and rabbit anti-mouse IgG HRP (1:2,000; ab6728) (both from Abcam) at 37 ̊C for 1 h. The immunocomplexes were visualized by enhanced chemiluminescence (Western Blotting Luminol reagent; Santa Cruz Biotechnology, Inc.).

Techniques: Cell Differentiation, Transfection, Control, Plasmid Preparation