Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: Evaluation of the expression of CD133 in cervical cancer stem cells. (A) Results of immunofluorescence, the FACS-sorted cervical cancer stem-like side population cells showed increased positivity to the CD133 stem cell surface protein. Magnification, ×400. (B) Quantification of the results of reverse transcription-quantitative polymerase chain reaction and (C) western blot analyses revealed that the relative mRNA and protein expression levels of CD133 were significantly elevated in the SP cells, compared with the MP cells. Equal concentrations of protein were loaded per lane. GAPDH was used as a loading control. (D) Relative expression levels of CD133 compared with GAPDH in SP and MP cells, from the western blot analysis. Data are presented as the mean ± standard error of the mean. ** P<0.01, vs. SP cells. FACS, fluorescence-activated cell sorting; SP, side population; MP, main population; CD, cluster of differentiation.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: Expressing, Immunofluorescence, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Fluorescence, FACS

Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: CD133 + SP cells show high levels of differentiation. (A) In vitro proliferation assay revealed that the proliferation rate of the CD133 + SP cells were significantly higher, compared with the MP cells. (B) Morphology of the CD133 + SP cells changed rapidly on day 5 and later developed filaments, which resembled fibroblast. Magnification, ×100. Data are presented as the mean ± standard error of the mean. * P<0.05 and ** P<0.01, between SP and MP cells. CD. cluster of differentiation; SP, side population; MP, main population; OD, optical density.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: In Vitro, Proliferation Assay

Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: CD133 + SP cells exhibit high self-renewal capacity. (A) A clone formation efficiency assay revealed that the total number of tumor spheres generated by the CD133 + SP cells were significantly higher, compared with the number generated by the MP cells. (B) Quantification of the results of reverse transcription-quantitative polymerase chain reaction analysis showed that the relative mRNA expression levels of Oct-4, EpCAM, Sox-2, Bmi-1 and Nestin were significantly upregulated in the CD133 + SP cells, compared with the MP cells. (C) Fluorescence microscopy revealed that the CD133 + SP cells exhibited more positive CD44 fluorescence and EpCAM stem cell proteins, whereas this fluorescence was not enriched in the MP cells. Magnification, ×400. Data are presented as the mean ± standard error of the mean. ** P<0.01, vs. SP. Oct-4, octamer-binding transcription factor-4; EpCam, epithelial cell adhesion molecule; Sox-1, (sex determining region Y)-box 2; Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site-1; CD. cluster of differentiation; SP, side population; MP, main population.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: Generated, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Fluorescence, Microscopy, Binding Assay, Virus

Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: CD133 + SP cells are multidrug and apoptosis resistant. (A) Comparison of cell survival rate between the CD133 + SP and MP cells following treatment with the DNA targeting drugs, 5-FU, oxaliplatin, cisplatin and paclitaxel. The SP cells showed increased resistance to these drugs, and had a higher survival rate following treatment, compared with the MP cells. (B) Number of CD133 + SP cells undergoing apoptosis were significantly lower, compared with the MP cells. (C) Quantification of results from reverse transcription-quantitative polymerase chain reaction analysis, showing that the relative mRNA expression levels of the ABC transporter gene, ABCG2, and the anti-apoptotic gene, Bcl-2, were significantly upregulated in the CD133 + SP cells. Data are presented as the mean ± standard error of the mean. ** P<0.01, between SP and MP cells. CD, cluster of differentiation; SP, side population; MP, main population; 5-FU, 5-fluorouracil; ABC, ATP-binding cassette.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: Comparison, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: Secretion of IL-4 in CD133 + SP cells. (A) Western blot analysis showed elevated secretion of IL-4 in CD133 + SP cells, compared with MP cells. (B) Relative expression levels of IL-4 compared with GAPDH in SP and MP cells, from the western blot analysis. (C) Cell viability of the CD133 + SP cells was significantly reduced following pretreatment with anti-IL-4 for 24 h. (D) Tumor spheres generated by CD133 + SP cells became more sensitive to apoptosis following pretreatment with anti-IL-4 for 24 h. Magnification, ×25. Data are presented as the mean ± standard error of the mean. * P<0.05 and ** P<0.01, between SP and MP cells. IL-4, interleukin-4; CD, cluster of differentiation; SP, side population; MP, main population; 5-FU, 5-fluorouracil.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: Western Blot, Expressing, Generated

Journal: Molecular Medicine Reports

Article Title: Production of interleukin-4 in CD133 + cervical cancer stem cells promotes resistance to apoptosis and initiates tumor growth

doi: 10.3892/mmr.2016.5195

Figure Lengend Snippet: CD133 + SP cells are highly tumorigenic and invasive. (A) CD133 + SP cells initiate tumor growth in NON/SCID mice significantly faster, compared with MP cells. (B) Immunohistochemistry showed that SP cell-derived tumor tissues were more positive to the CD133 stem cell protein. Magnification, ×250. (C) CD133 + SP cell invasiveness was measured using a Matrigel assay. The number of CD133 + SP cells, which invaded across the membrane were significantly higher, compared with the number of MP cells. Data are presented as the mean ± standard error of the mean. ** P<0.01, vs. SP. CD, cluster of differentiation; SP, side population; MP, main population.

Article Snippet: The cells were incubated with rabbit anti-CD133 fluorescein isothiocyanate (FITC)-conjugated antibody (dilution, 1:500; cat. no. orb15325; Biorbyt, Cambridge, UK) for 30 min.

Techniques: Immunohistochemistry, Derivative Assay, Matrigel Assay, Membrane

Journal: PLoS ONE

Article Title: CD166/ALCAM Expression Is Characteristic of Tumorigenicity and Invasive and Migratory Activities of Pancreatic Cancer Cells

doi: 10.1371/journal.pone.0107247

Figure Lengend Snippet: (A) qRT-PCR analysis of the mRNA levels of CD166 in SUIT-2 cells after RNA interference was performed at the indicated days post-transfection. Control (siControl) or CD166 silenced cells (siCD166) were analyzed by (B) colony formation assays at the indicated days post-transfection. (C) qRT-PCR analysis of EMT markers E-cadherin, N-cadherin, Zeb-1, and MMP2 in CD166+ and CD166- Panc-1 and SW1990 cells. (D) The relationships between expression of CD166 and CSC markers CD24, CD44, and CD133 in Panc-1 cells were analyzed by flow cytometry. Data represent the mean ± SD; *, p <0.05; NS, not significant.

Article Snippet: Cells from subconfluent monolayer cultures were suspended in phosphate-buffered saline (PBS) and incubated with monoclonal anti-human ALCAM-phycoerythrin (PE) (R&D Systems, Minneapolis, MN), anti-human CD24-fluorescein isothiocyanate (FITC) (eBioscience Inc, San Diego, CA), anti-human CD44-FITC (MBL, Nagoya, Japan), and monoclonal anti-human CD133-FITC (Ancell Corp, Bayport, MN) antibodies.

Techniques: Quantitative RT-PCR, Transfection, Expressing, Flow Cytometry

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a is overexpressed in prostate cancer and associated with VM formation mediated by cancer cell stemness and EMT. (A) The mRNA levels of Wnt5a were measured in a normal prostate epithelial cell line (RWPE-1) and prostate cancer cell lines (PC3, LNCaP, DU145) using quantitative polymerase chain reaction (qPCR), n = 3, * P ๤0.05 v.s. RWPE-1. (B, C) Western blot analysis was conducted to detect Wnt5a expression in the indicated cell lines, * P ๤0.05 v.s. RWPE-1, n = 3. (D-E) Immunohistochemical staining of WNT5A in tissues of BPH and PCa. D is a representative graph and E is a statistical graph, Original magnification, × 200; scale bar, 20 μm. * P ๤0.05 v.s. BPH, n = 50. (F) Representative images of CD31/PAS double staining and immunohistochemistry (IHC) staining were obtained from paraffin-embedded human prostate cancer specimens. The VM structure, indicated by the red arrow, exhibited a positive reaction to PAS staining and lacked CD31 staining. Additionally, the VM structure contained red blood cells, as observed in the magnified inset. On the other hand, the endothelium-dependent vessel, denoted by the black arrow, displayed positive staining for both CD31 and PAS, as observed in the magnified inset. Furthermore, the expression of Wnt5a was found to be higher in cases positive for VM (left) compared to cases negative for VM (right). Conversely, the marker for cellular epithelial characteristic, E-cadherin, was downregulated in cases positive for VM (left) compared to cases negative for VM (right). The expression levels of EMT-related protein (Vimentin) and CSC-associated protein (CD133) were found to be significantly higher in the VM positive cases (left) compared to the VM negative cases (right). Original magnification, × 200; scale bar, 20 μm, for insets, 10 μm.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemical staining, Staining, Double Staining, Immunohistochemistry, Marker

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: The correlation among VM, Wnt5a expression and EMT/CSCs-related proteins expressions in PCa tissues.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Expressing

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a functions as a regulator of VM formation mediated by EMT and stemness in PCa cell lines. (A) To investigate the role of Wnt5a in VM formation, DU145 cells were stably transfected with negative control shRNA lentivirus (stable-sh-Ctl) or shRNA lentivirus targeting Wnt5a (stable-shWnt5a). Subsequently, stable-shWnt5a cells were transiently transfected with either an empty vector plasmid (stable-shWnt5a+Vector) or a Wnt5a overexpression plasmid (stable-shWnt5a+Wnt5a-OV). The expression of Wnt5a in various cell groups was assessed using Western blot analysis. (B) The statistical results of gray values in (A) were presented as histogram. (C) LNCaP cells were transfected with either empty lentivirus (referred to as stable-Ctl) or lentivirus overexpressing Wnt5a (referred to as stable-Wnt5a). Subsequently, stable-Wnt5a cells were transiently transfected with negative control siRNA (referred to as stable-Wnt5a+NC) or siRNA specifically targeting Wnt5a (referred to as stable-Wnt5a+ si-Wnt5a). Western blot was applied to measure the expression of Wnt5a. (D) The statistic results of Western blot bands in (C). (E) The mRNA level of EMT-related genes (E-Cadherin, Vimentin), cell stemness-related genes (CD133, Nanog, Sox2, Oct4) and VM-related genes (VE-cadherin, MMP9) in DU145 cells from various groups were assessed using qPCR. (F, G) The expression levels of EMT-related proteins (E-Cadherin, Vimentin), cell stemness-related proteins (CD133, Nanog, Sox2, Oct4) and VM-related proteins (VE-cadherin, MMP9) in DU145 cells from different groups were determined through Western blot analysis. The grayscale statistical map of (F) was denoted as (G). (H-J) The relative expression levels of EMT-related genes, cell stemness-related genes and VM-related genes in LNCaP of different groups as indicated were measured using qPCR (H) and Western blot (I, J), (J) is the grayscale statistical map of (I). (K, L) Flow cytometric quantification was employed to determine the CD133-positive population in DU145 across different groups. The figures (K) and statistical chart (L) presented on the panel illustrate the CD133-positive population in DU145 across different groups. (M, N) Representative flow plots (M) and statistical chart (N) of CD133-positive in LNCaP of different groups are shown. (O, P) Representative images (O) and statistical chart (P) depict the tube formation in DU145. Original magnification, × 200; scale bar, 200 μm. (Q, R) Representative images (Q) and statistical chart (R) illustrate the tube formation in LNCaP. Original magnification, × 200; scale bar, 200 μm. * P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Stable Transfection, Transfection, Negative Control, shRNA, Plasmid Preparation, Over Expression, Expressing, Western Blot

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a facilitates VM formation mediated by EMT and cellular stemness in vivo. (A) The live imaging presented depicts the growth of tumors in mice at day 7, 14, and 21 subsequent to orthotopic-injection of DU145 cells. The experimental groups include stable-sh-Ctl, which represents DU145 cells with stable transfection of negative control shRNA lentivirus, and stable-shWnt5a, which represents DU145 cells with stable transfection of shRNA lentivirus targeting Wnt5a. (B) The statistical diagram of (A) is shown, * P <0.05 v.s. stable-sh-Ctl, n = 6. (C) The image corresponds to the tumor samples obtained on day 21. (D) Statistic graph of tumor size of (C), * P <0.05 v.s. stable-sh-Ctl, n = 6. (E) Live imaging map of tumor in mice at day 7, 14 and 21 after LNCaP as indicated orthotopic-injection. stable-Ctl, LNCaP with stable transfection of empty-vector lentivirus; stable-Wnt5a, LNCaP with stable transfection of Wnt5a overexpression lentivirus. (F) Shown is the statistical diagram of average chemiluminescence intensity in (E), * P <0.05 v.s. stable-Ctl, n = 6. (G) The image of the tumor samples at day 21. (H) Statistic graph of tumor size of (G), * P <0.05 v.s. stable-Ctl, n = 6. (I) Shown is CD31/PAS double staining for identification of VM structure and IHC staining for Wnt5a as well as VM-associated factors (VE-cadherin, MMP9) in tumor sections obtained from DU145 orthotopic-injected mouse. Original magnification, × 100; scale bar, 100 μm. (J-K) The statistical chart of (I), * P <0.05 v.s. stable-sh-Ctl, n = 6. (L) Images are the representatives for IHC staining of tumor sections obtained from DU145 orthotopic-injected mouse using antibodies against EMT-related proteins (E-Cadherin, Vimentin) and cell stemness-related proteins (CD133, Nanog, Sox2, Oct4). Original magnification, × 100; scale bar, 100 μm. (M) The graph of the statistical results of (L), * P <0.05 v.s. stable-sh-Ctl, n = 6. (N-R) Images are the representatives (N, Q) and statistical results (O, P, R) for CD31/PAS double staining and IHC staining of tumor sections obtained from LNCaP orthotopic-injected mouse. Original magnification, × 100; scale bar, 100 μm. * P <0.05 v.s. stable-Ctl, n = 6.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: In Vivo, Imaging, Injection, Stable Transfection, Negative Control, shRNA, Plasmid Preparation, Over Expression, Double Staining, Immunohistochemistry

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: The screening of the downstream signal pathway involved in Wnt5a-induced VM formation was conducted. (A) Western blot analysis was used to quantify the expression levels of Vimentin, CD133, Nanog, Sox2, OCT4, VE-cadherin, and MMP9 in stable-Ctl LNCaP and stable-Wnt5a LNCaP cells treated as indicated. SP600125, JNK inhibitor; U-73,122, PLC inhibitor; TFA, PKG inhibitor; LF3, β-Catenin inhibitor; HSD1590, ROCK inhibitor. (B, C) Representative flow plots (B) and statistical chart (C) of CD133-positive population in LNCaP of different groups as indicated are shown. (D) Images of VM formation in 3D-cultured LNCaP is shown. Original magnification, × 200; scale bar, 200 μm. (E) The number of completely formed tubes was quantitated in (D). * P <0.05 v.s. as indicated, n = 3. (F) The expression of genes as indicated in DU145 was detected by qPCR. * P <0.05 v.s. as indicated, n = 3. (G-H) Representative (G) and statistical (H) images of VM formation in 3D-cultured DU145 is shown. Original magnification, × 200; scale bar, 200 μm. P <0.05 v.s. as indicated, n = 3. (I) The expression of genes as indicated in PC3 was detected by qPCR. * P <0.05 v.s. as indicated, n = 3. (J-K) Representative images (J) and statistical graph (K) of VM formation in 3D-cultured PC3 is shown. Original magnification, × 200; scale bar, 200 μm. P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Western Blot, Expressing, Cell Culture

Journal: Translational Oncology

Article Title: Wnt5a promotes VM formation by modulating the stemness and EMT progression of prostate cancer cell

doi: 10.1016/j.tranon.2024.102155

Figure Lengend Snippet: Wnt5a activates the JNK/c-jun pathway and promotes VM formation in a JNK-dependent manner. (A) The phosphorylation levels of JNK and c-jun were detected by western blot in stable-sh-Ctl DU145 and stable-shWnt5a DU145 cells. (B) The histogram showing the statistical results of gray values in (A), * P <0.05 v.s. stable-sh-Ctl, n = 3. (C, D) Immunoblotting analysis of phosphorylation level of JNK and c-jun in stable-Ctl LNCaP and stable-Wnt5a LNCaP cells. (D) is the statistical results of gray values of (C), * P <0.05 v.s. stable-Ctl, n = 3. (E) Representative images of immunohistochemical staining of tumor sections obtained from DU145 orthotopic-injected mice using antibodies against p-JNK and p-c-jun are shown. Original magnification, × 100; scale bar, 100 μm. (F) The statistical results of (E), * P <0.05 v.s. stable-sh-Ctl, n = 6. (G) The phosphorylation level of JNK and c-jun in tumor sections obtained from LNCaP orthotopic-injected mouse were evaluated by IHC staining. Original magnification, × 100; scale bar, 100 μm. (H) The statistical results of (G), * P <0.05 v.s. stable-Ctl, n = 6. (I) The expression of Vimentin, CD133, Nanog, Sox2, VE-cadherin and MMP9 in stable-sh-Ctl DU145 and stable-shWnt5a DU145 cells treated as indicated was quantified by western Blot. Vector, Empty vector plasmid; JNK-OV, JNK overexpression plasmid. (J-O) These histograms showing the statistical results of gray values of (I), * P <0.05 v.s. as indicated, n = 3. (P, Q) Representative flow cytometry diagrams (P) and statistical graph (Q) of CD133-positive in DU145 of different groups as indicated are shown, * P <0.05 v.s. as indicated, n = 3. (R, S) Representative images (R) and statistical graph (S) of tube formation in DU145 treated as indicated, Original magnification, × 200; scale bar, 200 μm; * P <0.05 v.s. as indicated, n = 3.

Article Snippet: These suspensions were subsequently incubated with anti-CD133-FITC antibody (ZSGB-Bio, China) at room temperature for 1 h. An isotype IgG control was also included in the experimental setup.

Techniques: Western Blot, Immunohistochemical staining, Staining, Injection, Immunohistochemistry, Expressing, Plasmid Preparation, Over Expression, Flow Cytometry