Journal: Cell Death and Differentiation

Article Title: SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC

doi: 10.1038/cdd.2011.137

Figure Lengend Snippet: SOX2 knockdown alter WNT signaling through DKK1. ( a and b ) PCR array results. The expression of 88 frequently cited stem cell genes were compared between sh-control and sh-SOX2 cells. The expression of AXIN1 , KRT15 , and GJB2 increased by more than four-fold, and the expression of c-MYC , PARD6A , and CDH1 decreased by more than four-fold after SOX2 knockdown. ( c ) Real-time PCR analysis of WNT-related candidate genes. The expression of DKK1 was significantly decreased, but the expression of β-CATENIN was increased after SOX2 knockdown. * P <0.05; ** P <0.01. ( d ) The western blot indicates reduced phosphorylated β -CATENIN (p- β -CATENIN) expression and increased total β -CATENIN (total- β -CATENIN) expression after SOX2 knockdown in hUCB–MSCs. Protein levels were normalized to β -ACTIN using Image J analysis software. ( e ) The TOP/FOP Flash assay shows increased WNT signaling in sh-SOX2 cells. *** P <0.001

Article Snippet: The DKK1 recovery assay was conducted using a DKK1 plasmid vector (number SC303946; OriGene Technologies, Rockville, MD, USA) and a control vector (number PCMV6XL5; OriGene Technologies).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Software

Journal: Cell Death and Differentiation

Article Title: SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC

doi: 10.1038/cdd.2011.137

Figure Lengend Snippet: SOX2 directly binds to the DKK1 promoter region. ( a ) Sequence homology of region around DKK1 between mammals. Except for the exons of DKK1 , the region 1 kbps upstream of the transcriptional start site of DKK1 was highly conserved and contained an HMG homeodomain (−76 bps). ( b ) ChIP assay for the −76 bps region of DKK1 showed SOX2-specific binding. ( c ) Luciferase reporter assay. Luciferase activity and transfection efficiency were normalized to transfection of the SV40 promoter control vector (SV40 promoter). The human DKK1 promoter region from −974 bps to +119 bps around the DKK1 transcription start site, which contains an HMG-binding domain, was cloned into the luciferase enhancer vector ( DKK1 promoter). ( d ) The luciferase activity of sh-SOX2 was significantly decreased compared with that of the sh-control. *** P <0.001. ( e ) Site-directed mutagenesis, involving single (M1) and triple (M3) mutations, was performed in the HMG domain of the DKK1 promoter vector. ( f and g ) The M1 (also SOX2 binding site) mutation had no effect on the luciferase activity, but the M3 mutation significantly reduced the luciferase activity in both hUCB–MSCs and tera-1 cells. *** P <0.001

Article Snippet: The DKK1 recovery assay was conducted using a DKK1 plasmid vector (number SC303946; OriGene Technologies, Rockville, MD, USA) and a control vector (number PCMV6XL5; OriGene Technologies).

Techniques: Sequencing, Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Clone Assay, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC

doi: 10.1038/cdd.2011.137

Figure Lengend Snippet: Ectopic expression of DKK1 in SOX2-knockdown hUCB–MSCs reverted the differentiation malformation. ( a–c ) The adipogenic deformity of lentiviral-mediated SOX2-knockdown hUCB–MSCs was rescued after ectopic expression of DKK1, and this result was confirmed by morphology ( a ), oil red O elution ( b ), and marker gene analysis ( c ). The scale bar represents 100 μ m. ** P <0.01. ( d–f ) The ability of hUCB–MSCs to undergo osteogenic differentiation upon SOX2 knockdown by lentivirus was decreased after the ectopic expression of DKK1, and this result was confirmed by morphology ( d ), alizarin red S elution ( e ), and marker gene analysis ( f ). The scale bar represents 100 μ m. *** P <0.001. ( g and h ) The ability of hUCB–MSCs to undergo chondrogenic differentiation upon SOX2 knockdown by lentivirus was decreased by the ectopic expression of DKK1, and this result was confirmed by pellet size ( g ) and marker gene analysis ( h ). The scale bar represents 100 μ m

Article Snippet: The DKK1 recovery assay was conducted using a DKK1 plasmid vector (number SC303946; OriGene Technologies, Rockville, MD, USA) and a control vector (number PCMV6XL5; OriGene Technologies).

Techniques: Expressing, Marker

Journal: Cell Death and Differentiation

Article Title: SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC

doi: 10.1038/cdd.2011.137

Figure Lengend Snippet: Ectopic expression of DKK1 and WNT ligands cannot recover the proliferation defect caused by SOX2 knockdown in hUCB–MSCs. ( a and b ) Proliferation of hUCB–MSCs with lentiviral SOX2 knockdown did not increase after DKK1 treatment, as confirmed by the MTT assays and FACS analysis. * P <0.05; ** P <0.01; *** P <0.001. ( c and d ) Treatment with WNT3A and WNT5A could not recover the proliferation defect in SOX2-knockdown hUCB–MSCs. ** P <0.01; *** P <0.001

Article Snippet: The DKK1 recovery assay was conducted using a DKK1 plasmid vector (number SC303946; OriGene Technologies, Rockville, MD, USA) and a control vector (number PCMV6XL5; OriGene Technologies).

Techniques: Expressing

Journal: Cell Death and Differentiation

Article Title: SOX2 has a crucial role in the lineage determination and proliferation of mesenchymal stem cells through Dickkopf-1 and c-MYC

doi: 10.1038/cdd.2011.137

Figure Lengend Snippet: Schematic diagram of proposed mechanism for regulatory roles of SOX2 in hMSCs. SOX2 bound DKK1 promoter region and induced DKK1 transcription. DKK1 regulates differentiation of hMSCs, but could not control proliferation. Proliferation was influenced by c-MYC, which was controlled by SOX2

Article Snippet: The DKK1 recovery assay was conducted using a DKK1 plasmid vector (number SC303946; OriGene Technologies, Rockville, MD, USA) and a control vector (number PCMV6XL5; OriGene Technologies).

Techniques:

Journal: International Journal of Biological Sciences

Article Title: Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

doi: 10.7150/ijbs.52097

Figure Lengend Snippet: DKK1 expression levels in the CSIOVDB database. Gene expression profiles of DKK1 by the ovarian cancer disease state (A), FIGO stage (B), FIGO grade (C) and survival curve (D). Abbreviation: OSE, ovarian surface epithelium; FTE, fallopian tube epithelium; Mets, metastasis; Error bar is the median ± quantile. Kaplan-Meier analyses were conducted to estimate overall survival and disease-free survival. *p<0.05, ****p<0.0001.

Article Snippet: Determination of DKK1 in culture medium was performed using a commercial ELISA kit (EK0867) (Boster, China) according to the manufacturer's instructions.

Techniques: Expressing, Gene Expression

Journal: International Journal of Biological Sciences

Article Title: Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

doi: 10.7150/ijbs.52097

Figure Lengend Snippet: CA125 downregulates DKK1 expression in ovarian cancer cells. qPCR (A-B) , ELISA (C-D) , and Western blot (E-F) analysis of DKK1 mRNA levels in A2780 and OVCAR3 cells treated with CA125 at 0, 0.2. and 0.4 µg/mL for 48 h. The results represent the mean ± SD. *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: Determination of DKK1 in culture medium was performed using a commercial ELISA kit (EK0867) (Boster, China) according to the manufacturer's instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

doi: 10.7150/ijbs.52097

Figure Lengend Snippet: CA125 induced ovarian cancer cell migration by attenuating DKK1 expression. OVCAR3 cells (A) transfected with pEGFP-N1 vector and pEGFP-N1-DKK1 and A2780 (B) transfected with negative control (NC) and siDKK1 were subjected to Transwell migration assays and treated for 48 h with CA125 (scale bars=100 µm). (C) (D) Cell counts across the Transwell membrane. The statistical analyses were conducted with the number of invaded cells (indicated by red arrows). The results represent the mean ± SD. *p<0.05, **p<0.01, *** p<0.001.

Article Snippet: Determination of DKK1 in culture medium was performed using a commercial ELISA kit (EK0867) (Boster, China) according to the manufacturer's instructions.

Techniques: Migration, Expressing, Transfection, Plasmid Preparation, Negative Control, Membrane

Journal: International Journal of Biological Sciences

Article Title: Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

doi: 10.7150/ijbs.52097

Figure Lengend Snippet: CA125 reduces DKK1 expression to activate the SGK3/FOXO3 pathway. (A) (C) (E) Western blots were performed to evaluate the expression of Akt, p-Akt, PI3K, and SGK3 in total cell lysates (TCL) and FOXO3 in cytoplasmic/nuclear protein extracts. GAPDH, Tubulin-β and PCNA were used as the experimental controls. (B) (D) (F) Relative protein expression of blots was quantified using densitometric analysis. The results represent the mean ± SD. There was no statistically significant difference.

Article Snippet: Determination of DKK1 in culture medium was performed using a commercial ELISA kit (EK0867) (Boster, China) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Free CA125 promotes ovarian cancer cell migration and tumor metastasis by binding Mesothelin to reduce DKK1 expression and activate the SGK3/FOXO3 pathway

doi: 10.7150/ijbs.52097

Figure Lengend Snippet: Anti-MSLN blockade of CA125 reduced the decrease in DKK1 and initiated apoptosis. (A) Expression of DKK1 was determined by ELISAs. (B) The expression levels of Tubulin-β in total cell lysates were determined by Western blots. (C-D) Flow cytometric analysis of OVCAR3 cells treated with and without anti-MSLN or CA125. Statistical analysis of the gated cells and representative images are shown. The results represent the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns, not significant.

Article Snippet: Determination of DKK1 in culture medium was performed using a commercial ELISA kit (EK0867) (Boster, China) according to the manufacturer's instructions.

Techniques: Expressing, Western Blot