Journal: Oncology Reports

Article Title: FGD5-AS1 is an oncogenic lncRNA in pancreatic cancer and regulates the Wnt/β-catenin signaling pathway via miR-577

doi: 10.3892/or.2021.8232

Figure Lengend Snippet: miR-577 targets LRP6 and β-catenin. (A) A dual-luciferase reporter assay was utilized to validate the binding sites between miR-577 and LRP6 3′UTR. (B) A dual-luciferase reporter assay was utilized to validate the binding sites between miR-577 and β-catenin 3′UTR. (C) Reverse transcription-quantitative PCR was employed to detect LRP6 and β-catenin mRNA expression levels in SW1990 cells transfected with miR-577 mimics. (D) A TOPFlash luciferase reporter assay was performed to detect the effects of miR-577 on the activity of the Wnt/β-catenin signaling. (E and F) Pearson's correlation analysis was utilized for the correlations between (E) FGD5-AS1 and LRP6 and between (F) FGD5-AS1 and β-catenin in pancreatic cancer tissues. (G and H) Pearson's correlation analysis was utilized for the correlations between (G) miR-577 and LRP6 and between (H) miR-577 and β-catenin in pancreatic cancer tissues. All of the experiments were performed in triplicate. **P<0.01 and ***P<0.001. LRP6, low-density lipoprotein receptor-related protein 6; miR, microRNA; FGD5-AS1, FGD5 antisense RNA 1; UTR, untranslated region.

Article Snippet: To determine the activity of Wnt/β-catenin pathway, the TOPflash and FOPflash system (Upstate Biotechnology, Inc.) was used.

Techniques: Luciferase, Reporter Assay, Binding Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Transfection, Activity Assay

Journal:

Article Title: c-Jun N-terminal kinase 1 interacts with and negatively regulates Wnt/?-catenin signaling through GSK3? pathway

doi: 10.1093/carcin/bgn239

Figure Lengend Snippet: Loss of JNK1 promoted β-catenin protein expression and β-catenin-mediated transcriptional activity of TCF. (A) β-Catenin protein expression was upregulated in JNK1−/− MEFs. Both MEFs were lysed and subjected to immunoblotting analysis detecting the expression of β-catenin and JNK1. β-Actin was used as loading control. (B) β-Catenin–TCF4 reporter activity was upregulated in JNK1−/− MEFs. JNK1+/+ and JNK1−/− MEFs were cotransfected with Renilla and a construct of TOPFLASH or FOPFLASH. Twenty-four hours after transfection, cells were treated with Wnt3a condition medium of 1:4 dilution for 24 h and harvested for luciferase activity assay.

Article Snippet: TCF-4 reporter plasmid TOPFLASH and the control inactive reporter FOPFLASH were from Upstate Biotechnology (Lake Placid, NY).

Techniques: Expressing, Activity Assay, Western Blot, Construct, Transfection, Luciferase

Journal:

Article Title: c-Jun N-terminal kinase 1 interacts with and negatively regulates Wnt/?-catenin signaling through GSK3? pathway

doi: 10.1093/carcin/bgn239

Figure Lengend Snippet: Active JNK1 downregulated β-catenin expression and inhibited its transcriptional activity. (A) Active JNK1 reduced β-catenin protein level in HEK cell line HEK293T. HEK293T cells were cotransfected with pcDNA3-Flag-MKK7-JNK1 and pcDNA3-HA-β-catenin. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK and p-c-Jun. β-Actin served as loading control. The density of band was quantified and the expression of β-catenin was normalized to β-actin. (B) Active JNK1 inhibited β-catenin-mediated transcriptional activity of TCF4. HEK293T cells were cotransfected with pcDNA3-Flag-MKK7-JNK1, pcDNA3-HA-β-catenin, TOPFLASH or FOPFLASH and a Renilla construct. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± SD for triplicate samples. Similar experiments were done in human colon cancer cell line SW480 (C and D). (E) Activated exogenous JNK1 reduced β-catenin protein level in a dose-dependent manner. HEK293T cells were cotransfected with pcDNA3-HA-β-catenin and different amounts of pcDNA3-Flag-MKK7-JNK1, as indicated. The protein was obtained for immunoblotting analysis to detect the alterations of HA-β-catenin and p-JNK. β-Actin served as loading control. (F) Activated exogenous JNK1 inhibited β-catenin-mediated transcriptional activity of TCF in a dose-dependent manner. HEK293T cells were cotransfected with pcDNA3-HA-β-catenin, TOPFLASH, Renilla, along with different amounts of pcDNA3-Flag-MKK7-JNK1, as indicated. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± SD for triplicate samples.

Article Snippet: TCF-4 reporter plasmid TOPFLASH and the control inactive reporter FOPFLASH were from Upstate Biotechnology (Lake Placid, NY).

Techniques: Expressing, Activity Assay, Transfection, Western Blot, Construct, Luciferase

Journal: Arthritis Research & Therapy

Article Title: Low-density lipoprotein receptor–related protein 5 governs Wnt-mediated osteoarthritic cartilage destruction

doi: 10.1186/ar4466

Figure Lengend Snippet: Catabolic regulation of LRP5 mediates via β-catenin-Tcf/Lef signaling. (A) Chondrocytes were transfected with 1 μg of empty vector (EV) or pSPORT6- Lrp5 plus the TOPflash or FOPflash reporter constructs. After 24 hours, transcriptional activation by β-catenin was determined by luciferase reporter gene assays ( n = 7 independent experiments). (B) Chondrocytes obtained from wild-type (WT) and Lrp5 -/- mice were treated with 1 ng/ml interleukin 1β (IL-1β), 50 ng/ml Wnt3a or 500 ng/ml Wnt7a, and transcriptional activation by β-catenin was evaluated by luciferase reporter gene assays ( n = 6). Values are expressed as means ± SEM (* P < 0.005). (C) β-catenin and LRP5 expression levels in cartilage after sham operation or destabilization of the medial meniscus (DMM) surgery were determined by immunofluorescence microscopy. 4′,6-diamidino-2-phenylindole (DAPI) staining was used for visualization of nuclei. Scale bar: 20 μm. (D) β-catenin, matrix metalloproteinase 3 (MMP3) and MMP13 expression levels in undamaged (Normal) and damaged (osteoarthritis; OA) human osteoarthritic cartilage were examined by immunostaining. (E) and (F) β-catenin, MMP3 and MMP13 expression levels in spontaneous (aging-induced) osteoarthritic cartilage and DMM-induced osteoarthritic cartilage from Lrp5 -/- mice and their WT littermates were examined by immunohistochemistry. Scale bar: 50 μm.

Article Snippet: Chondrocytes were transfected with 1 μg of reporter gene (TOPflash) or control gene (FOPflash) (both from Upstate Biotechnology, Lake Placid, NY, USA) and 1 μg of pCMV - β-galactosidase using Lipofectamine 2000.

Techniques: Transfection, Plasmid Preparation, Construct, Activation Assay, Luciferase, Expressing, Immunofluorescence, Microscopy, Staining, Immunostaining, Immunohistochemistry

Journal: BMC Cancer

Article Title: Siah1 proteins enhance radiosensitivity of human breast cancer cells

doi: 10.1186/1471-2407-10-403

Figure Lengend Snippet: Effects of Siah1 on Tcf/Lef regulated transcription activity in SKBR3 and MCF-7 cells . A: Transfected cells were cotransfected with Topflash or Fopflash plasmid, the Renilla luciferase reporter plasmid (pRL-TK) as an internal control and the indicated expression plasmids. Luciferase activity was measured at 24 h after transfection and plotted after normalizing with respect to the Renilla luciferase activity. Each experiment was performed at least three times. Columns, mean; bars, SD; *, p < 0.05. B; Functional inhibition of Siah-1 ubiquitin-ligase by siRNA increased TCF/Lef transcriptional activity in MCF-7 cells at 24 hours after transfection. Each experiment was performed three times. Columns, mean; bars, SD; *, Siah1 siRNA vs. both untreated and control siRNA p < 0.05.

Article Snippet: The Tcf/Lef-responsive luciferase reporter gene (Topflash), the negative control with mutated Tcf/Lef binding site (Fopflash), and the Renilla luciferase reporter plasmid (pRL-TK) as an internal control were obtained from Upstate Biotechnology, USA.

Techniques: Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Functional Assay, Inhibition