Journal: BioMed Research International

Article Title: The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening

doi: 10.1155/2014/978984

Figure Lengend Snippet: Primers used for real-time RT-PCR.

Article Snippet: After being blocked by 5% nonfat milk, the membranes were incubated with goat anti-PinX1 antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, USA) and HRP conjugated rabbit anti-goat IgG (1 : 5000, Multisciences, Hangzhou, China) for the detection of PinX1 protein. β -actin was detected by using the mouse anti- β -actin antibody (1 : 2000, Multisciences, Hangzhou, China) and HRP conjugated goat anti-mouse IgG antibody (1 : 5000, Multisciences, Hangzhou, China).

Techniques:

Journal: BioMed Research International

Article Title: The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening

doi: 10.1155/2014/978984

Figure Lengend Snippet: qRT-PCR and western blotting analysis of PinX1 expression in PinX1 overexpressed and knocked-down breast cell lines. (a) Fold changes (2 −ΔΔCt values) by qRT-PCR showed increased expression of PinX1 mRNA in the pcDNA3.1-PinX1 transfected breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 when compared with their counterpart untransfected cells and empty vector transfected control cells. Expression levels were normalized for GAPDH. (b) Western blotting indicated upregulation of PinX1 protein in the pcDNA3.1-PinX1 transfected breast cancer cell lines MCF-7, MDA-MB-231, and SK-BR-3 in comparison with untransfected cells and empty vector transfected control cells. (c) Fold changes (2 −ΔΔCt values) by qRT-PCR showed decreased expression of PinX1 mRNA in the PinX1 siRNA fragments transfected MCF-10A cells, when compared with the untransfected cells and siRNA NC transfected control cells. (d) Western blotting indicated downregulation of PinX1 protein in the PinX1 siRNA fragments transfected MCF-10A cells in comparison with untransfected cells and siRNA NC transfected control cells.

Article Snippet: After being blocked by 5% nonfat milk, the membranes were incubated with goat anti-PinX1 antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, USA) and HRP conjugated rabbit anti-goat IgG (1 : 5000, Multisciences, Hangzhou, China) for the detection of PinX1 protein. β -actin was detected by using the mouse anti- β -actin antibody (1 : 2000, Multisciences, Hangzhou, China) and HRP conjugated goat anti-mouse IgG antibody (1 : 5000, Multisciences, Hangzhou, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Comparison

Journal: BioMed Research International

Article Title: The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening

doi: 10.1155/2014/978984

Figure Lengend Snippet: Growth control of breast cell lines by PinX1 overexpression and knockdown. (a) MTT assay showed a lower growth rate in the pcDNA3.1-PinX1 transfected MCF-7 cells than the untransfected and vector transfected control cells. (b) MTT assay showed a higher growth rate in the PinX1 siRNA3 tranfected MCF-10A cell line than the untransfected and siRNA NC transfected control cells. (c) Colorimetric focus-formation assay showed pcDNA3.1-PinX1 stable transfected MCF-7 cells had a lower focus counting than the empty vector stably tranfected control cells. (d), (e), and (f) Flow cytometry analysis indicated a G0/G1 phase arrest and S phase inhibition in the pcDNA3.1-PinX1 transfected MCF-7 cells compared to the untransfected and vector transfected control cells. (g), (h), and (i) Flow cytometry analysis indicated a decreased G0/G1 phase and increased S phase rate in the PinX1 knockdown MCF-10A cells compared to the untransfected and siRNA NC transfected control cells.

Article Snippet: After being blocked by 5% nonfat milk, the membranes were incubated with goat anti-PinX1 antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, USA) and HRP conjugated rabbit anti-goat IgG (1 : 5000, Multisciences, Hangzhou, China) for the detection of PinX1 protein. β -actin was detected by using the mouse anti- β -actin antibody (1 : 2000, Multisciences, Hangzhou, China) and HRP conjugated goat anti-mouse IgG antibody (1 : 5000, Multisciences, Hangzhou, China).

Techniques: Control, Over Expression, Knockdown, MTT Assay, Transfection, Plasmid Preparation, Tube Formation Assay, Stable Transfection, Flow Cytometry, Inhibition

Journal: BioMed Research International

Article Title: The Role of PinX1 in Growth Control of Breast Cancer Cells and Its Potential Molecular Mechanism by mRNA and lncRNA Expression Profiles Screening

doi: 10.1155/2014/978984

Figure Lengend Snippet: Microarray screening of the mRNA and lncRNA expression profile alterations in PinX1 overexpressed MCF-7 cells and qRT-PCR validation. (a), (b), (c), and (d) Heat maps and scatterplots of the distinguishable mRNA and lncRNA expression profiles between the pcDNA3.1-PinX1 group and the empty vector group of MCF-7 cells. Hierarchical clustering was performed and the results were displayed as a heat map, in which red denotes high relative expression levels and blue denotes low relative expression levels. (e) qRT-PCR validation of the microarray data in different breast cell lines. The expression fold change of the pcDNA3.1-PinX1 group versus the empty vector group of MCF-7 cells was verified by calculating the 2 −ΔΔCt of real-time RT-PCR results. The result showed that the fold change in expression by qRT-PCR was mainly consistent with the microarray data.

Article Snippet: After being blocked by 5% nonfat milk, the membranes were incubated with goat anti-PinX1 antibody (1 : 500, Santa Cruz Biotechnology, Santa Cruz, USA) and HRP conjugated rabbit anti-goat IgG (1 : 5000, Multisciences, Hangzhou, China) for the detection of PinX1 protein. β -actin was detected by using the mouse anti- β -actin antibody (1 : 2000, Multisciences, Hangzhou, China) and HRP conjugated goat anti-mouse IgG antibody (1 : 5000, Multisciences, Hangzhou, China).

Techniques: Microarray, Expressing, Quantitative RT-PCR, Biomarker Discovery, Plasmid Preparation

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: PinX1 staining and clinicopathological characteristics of 405 breast cancer patients

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Staining

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: Expression of PinX1 in breast cancer tissues and associated with 5-year overall and disease-specific survival in breast cancer patients. (A) Negative and positive staining in breast cancer tissue. Top panel, magnification × 200; bottom panel, magnification × 400. (B) Low PinX1 expression correlated with a poorer 5-year overall cumulative survival for 236 breast cancer patients ( P = 0.010, log-rank test). (C) Low PinX1 expression correlated with a poorer 5-year disease-specific cumulative survival for 208 breast cancer patients ( P = 0.003, log-rank test). Cum. indicates cumulative.

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Expressing, Staining

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: Univariate Cox proportional regression analysis on 5-year overall and disease-specific survival of 405 breast cancer patients

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques:

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: Multivariate Cox regression analysis on 5-year overall and disease-specific survival of 405 breast cancer patients

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques:

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: PinX1 inhibits migration and invasion of human breast cancer cells. (A) Western blot analysis of the relative protein level of PinX1 in PinX1 knockdown (siPinX1) and control siRNA (siCtrl) groups for both MDA-MB-231 and BT-549 cell lines. (B) Western blot analysis of the relative protein level of PinX1 in PinX1 overexpression (PinX1 OE ) and control vector (Vector) groups for both MDA-MB-231 and BT-549 cell lines. (C) and (E) PinX1 knockdown significantly inhibited migration and invasion abilities of MDA-MB-231 and BT-549 cells. (D) and (F) PinX1 overexpression significantly inhibited migration and invasion abilities of MDA-MB-231 and BT-549 cells. All experiments were carried out in triplicate. Data are shown as means ± SD. ***, P < 0.001.

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Migration, Western Blot, Knockdown, Control, Over Expression, Plasmid Preparation

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: PinX1 inhibits migration and invasion of breast cancer cells by suppressing MMP-9 expression and activity. (A) Western blot analysis of the relative protein levels of PinX1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in PinX1 siRNA and control siRNA group for both MDAMB-231 and BT-549 cell lines. (B) Western blot analysis of the relative protein levels of PinX1, MMP-2, MMP-9, TIMP-1 and TIMP-2 in PinX1 OE and Vector groups for both MDA-MB-231 and BT-549 cell lines. (C) Top panel, gelatin zymography analysis of the relative enzyme activities of MMP-9 in PinX1 knockdown and control siRNA group for both MDA-MB-231 and BT-549 cell lines. Bottom panel, gelatin zymography analysis of the relative enzyme activities of MMP-9 in PinX1 OE and Vector groups for both MDA-MB-231 and BT-549 cell lines. (D) Western blotting of PinX1 and MMP-9 in there groups of control siRNA, PinX1 siRNA, and PinX1 siRNA co-treated with MMP-9 inhibitor I for both MDA-MB-231 and BT-549 cell lines. (E) Gelatin zymography analysis of MMP-9 in there groups of control siRNA, PinX1 siRNA, and PinX1 siRNA co-treated with MMP-9 inhibitor I for both MDA-MB-231 and BT-549 cell lines. (F) and (G) The increased abilities of migration and invasion regulated by PinX1 knockdown in breast cancer cells was blocked by MMP-9 inhibitor I. All experiments were carried out in triplicate. Histograms represent means ± SD. ***, P < 0.001.

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Migration, Expressing, Activity Assay, Western Blot, Control, Plasmid Preparation, Zymography, Knockdown

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: PinX1 suppress MMP-9 expression via NF-κB-dependent transcription. (A) and (B) Western blot analysis of the relative protein levels of PinX1, MMP-9 and NF-κB-p65 in four groups of control, PinX1 OE , PinX1 KD and PinX1 KD co-treated with NF-κB-p65 siRNA for both MDA-MB-231 and BT-549 stable cell lines. The NF-κB specific siRNA dramatically prevented the up-regulation of MMP-9 expression induced by PinX1 knockdown. (C) and (D) The increased ability of migration and invasion regulated by PinX1 knockdown was suppressed by NF-κB-p65 siRNA in both MDA-MB-231 and BT-549 stable cell lines. All experiments were carried out in triplicate. Histograms represent means ± SD. ***, P < 0.001.

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Expressing, Western Blot, Control, Stable Transfection, Knockdown, Migration

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: PinX1 inhibits breast cancer cells metastasis in vivo. (A) Left panel, Western blotting of PinX1 in there groups of Ctrl-MDA-MB-231 cell lines, PinX1 OE -MDA-MB-231 cell lines and PinX1 KD -MDA-MB-231 cell lines, which was selected with puromycin for 2 weeks after lentivirus infection. Right panel, without puromycin selection for 2 months, PinX1 expression levels were not changed in MDA-MB-231 stable cell lines. (B) H&E staining sections of lungs 2 months after injection of PinX1 KD -MDA-MB-231 cell lines in BALB/c nude mouse through tail vein. Left panel, magnification × 100; right panel, magnification × 400. (C) Representative images of 10% buffered formalin fixed lungs with metastatic nodules 2 months after injection of Ctrl, PinX1 OE and PinX1 KD MDA-MB-231 stable cell lines. Arrows indicate metastatic nodules. (D) The number of lung metastatic nodules was counted under a dissecting microscope. Compared with the PinX1 OE group, a statistically dramatic increase in the number of the lung metastases was seen in PinX1 KD group, and these two groups also had significant diversity compared with Ctrl group respectively. Data are displayed with means ± SD from 10 mice in each group. ***, P < 0.001.

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: In Vivo, Western Blot, Infection, Selection, Expressing, Stable Transfection, Staining, Injection, Microscopy

Journal: Molecular Cancer

Article Title: PinX1 inhibits the invasion and metastasis of human breast cancer via suppressing NF-κB/MMP-9 signaling pathway

doi: 10.1186/s12943-015-0332-2

Figure Lengend Snippet: A Model of PinX1 suppresses MMP-9 expression via NF-κB signaling pathway. We speculate that PinX1 can suppress the expression of MMP-9 owing to the inhibition of transcriptional activity of NF-κB p65 proteins by direct protein-protein interaction with its G-patch domain which could combine with the C-terminus of the NF-κB-repression factor (NRF).

Article Snippet: Then subsequently the TMA slides were incubated with a polyclonal rabbit anti-PinX1 antibody (1:50 dilution; Novus Biologicals, Littleton, CO, USA) at 4°C overnight, and 3, 3′-diaminobenzidine (DAB; Zhongshan Biotech, Beijing, China) was used to produce a brown precipitate.

Techniques: Expressing, Inhibition, Activity Assay