stable ptaf knockdown skov3 cell lines  (Biowit Technologies)

Journal: Molecular Cancer

Article Title: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

doi: 10.1186/s12943-018-0844-7

Figure Lengend Snippet: miR-25 knockdown promotes OvCa cell migration. a The expression of miR-25 in OvCa cells after treatment with 10 ng/ml TGF-β1 for 48 h. b The mRNA levels of EMT-related markers in SKOV3 cells transfected with a miR-25 inhibitor (AMO-25) or a negative control (NC). c Western blotting analysis of epithelial and mesenchymal markers in SKOV3 cells transfected with AMO-25. Wound-healing ( d ) and migration assays ( e ) were used to determine the effect of miR-25 inhibition on OvCa cell migration. n = 5 independent experiments. * P < 0.05, ** p < 0.01 vs. NC

Article Snippet: In accordance with the manufacturer’s instructions, stable PTAF knockdown or overexpression SKOV3 cell lines and respective control cell lines were generated using lentiviral vectors carrying either sh-PTAF, a PTAF overexpression construct, or the respective negative controls (Biowit Technology, Shenzhen, China).

Techniques: Knockdown, Migration, Expressing, Transfection, Negative Control, Western Blot, Inhibition

Journal: Molecular Cancer

Article Title: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

doi: 10.1186/s12943-018-0844-7

Figure Lengend Snippet: Forced expression of miR-25 blunts TGF-β1-induced EMT and migration in OvCa cells. qRT-PCR ( a ) and western blot ( b ) analyses showed the inhibitory effect of miR-25 on EMT in SKOV3 cells treated with TGF-β1. A wound-healing assay displayed the inhibitory effects of miR-25 on TGF-β1-induced migration in SKOV3 cells ( c ) and in A2780 cells ( d ). A migration assay showed that miR-25 attenuated TGF-β1-induced migration in SKOV3 cells ( e ) and A2780 cells ( f ). n = 5 independent experiments. * P < 0.05, ** P < 0.01

Article Snippet: In accordance with the manufacturer’s instructions, stable PTAF knockdown or overexpression SKOV3 cell lines and respective control cell lines were generated using lentiviral vectors carrying either sh-PTAF, a PTAF overexpression construct, or the respective negative controls (Biowit Technology, Shenzhen, China).

Techniques: Expressing, Migration, Quantitative RT-PCR, Western Blot, Wound Healing Assay

Journal: Molecular Cancer

Article Title: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

doi: 10.1186/s12943-018-0844-7

Figure Lengend Snippet: PTAF regulates the expression and activity of miR-25. a PTAF contains a sequence domain complementary to the seed motif of miR-25. b Up-regulation of PTAF in OvCa cells treated with 10 ng/ml TGF-β1 for 48 h. n = 6 independent experiments. ** P < 0.01 vs. Control. c - d Overexpression of PTAF inhibited the expression of miR-25 in SKOV3 cells. n = 6 independent experiments. ** P < 0.01 vs. pcDNA3.1. e - f Silencing of PTAF using a specific shRNA up-regulated miR-25 in SKOV3 cells. n = 6 independent experiments. * P < 0.05, ** P < 0.01 vs. sh-Scramble. g - h PTAF binds to miR-25 and regulates its activity. * P < 0.05, ** P < 0.01. i PTAF directly binding to miR-25. SKOV3 cells were transfected with biotin-tagged miR-Ctrl (Bio-miR-Ctrl) or biotin-tagged miR-25 (Bio-miR-25). Forty-eight hours after transfection, the cells were harvested for a biotin-based pull-down assay. PTAF expression levels were analyzed by qRT-PCR. ** P < 0.01. j Luciferase reporter activities of chimeric vectors carrying the luciferase gene and a fragment of PTAF containing the wild-type (WT) binding site or a mutated binding site for miR-25. ** P < 0.01. k - l qRT-PCR were used to examine the expression of PTAF in SKOV3 cells after overexpression or knockdown of miR-25. * P < 0.05, ** P < 0.01. n = 6 independent experiments

Article Snippet: In accordance with the manufacturer’s instructions, stable PTAF knockdown or overexpression SKOV3 cell lines and respective control cell lines were generated using lentiviral vectors carrying either sh-PTAF, a PTAF overexpression construct, or the respective negative controls (Biowit Technology, Shenzhen, China).

Techniques: Expressing, Activity Assay, Sequencing, Control, Over Expression, shRNA, Binding Assay, Transfection, Pull Down Assay, Quantitative RT-PCR, Luciferase, Knockdown

Journal: Molecular Cancer

Article Title: Systematic analyses reveal long non-coding RNA (PTAF)-mediated promotion of EMT and invasion-metastasis in serous ovarian cancer

doi: 10.1186/s12943-018-0844-7

Figure Lengend Snippet: PTAF silencing inhibits tumor progression in an orthotopic mouse model of OvCa. a Representative bioluminescence images of mice injected intraperitoneally with SKOV3 OvCa cells expressing either PTAF or empty pcDNA3.1 ( n = 10 in each group). b More tumor masses (yellow arrows) were formed by SKOV3-PTAF cells than by SKOV3-pcDNA3.1 cells. c Mice injected with SKOV3-PTAF cells showed liver metastases. d Representative images of tumor nodules and quantification of tumor nodules and tumor weights in mice injected intraperitoneally with SKOV3 OvCa cells expressing either PTAF or empty pcDNA3.1 ( n = 10 in each group). ** P < 0.01 vs. pcDNA3.1. e SKOV3-ip tumor samples from mice injected with cells expressing PTAF or empty pcDNA3.1 were immunohistochemically stained for E-cadherin and SNAI2. * P < 0.05 vs. pcDNA3.1. f Representative bioluminescence images in sh-Scramble- and sh-PTAF-treated mice ( n = 10 in each group) that were injected intraperitoneally with SKOV3 OvCa cells. g Fewer tumor masses (yellow arrows) were formed in the mice treated with sh-PTAF than in those treated with sh-Scramble. h Mice injected with sh-Scramble showed liver metastases, whereas very little metastasis was observed in mice injected with sh-PTAF. i Representative images of tumor nodules and quantification of tumor nodules and tumor weights in sh-Scramble- and sh-PTAF-treated mice injected with SKOV3 cells ( n = 10 for each group). ** P < 0.01 vs. sh-Scramble. j SKOV3-ip tumor samples from sh-Scramble- and sh-PTAF-treated mice were immunohistochemically stained for E-cadherin and SNAI2. * P < 0.05 vs. sh-Scramble. Scale bars, 100X = 100 μm; 400X = 20 μm

Article Snippet: In accordance with the manufacturer’s instructions, stable PTAF knockdown or overexpression SKOV3 cell lines and respective control cell lines were generated using lentiviral vectors carrying either sh-PTAF, a PTAF overexpression construct, or the respective negative controls (Biowit Technology, Shenzhen, China).

Techniques: Injection, Expressing, Staining