Journal: Reproduction (Cambridge, England)

Article Title: Estradiol promotes cells invasion by activating β-catenin signaling pathway in endometriosis

doi: 10.1530/REP-15-0371

Figure Lengend Snippet: (A, B, C and D) Effects of E 2 and E 2 +ICI on the β-catenin expression. (A and C) Dose-dependence by the stimulation with E 2 . HESCs were cultured in a phenol red-free DMEM/F12 for 24 h and incubated with various concentrations of E 2 and E 2 +ICI for 24 h. (B and D) Time course by the stimulation with E 2 . Cells were cultured in a phenol red-free DMEM/F12 for 24 h and treated with E 2 (10 −8 mol/l), E 2 +ICI for the indicated times (0, 12, 24, and 48 h). β-catenin mRNA was extracted by TRIzol and examined by RT-PCR (A and B). Protein samples were separated by 10% SDS–PAGE and subjected to western blot analysis for total β-catenin (C and D). (E) Effects of E 2 on the nuclear localization of β-catenin in HESCs. Cells were stimulated with DMSO or E 2 (10 −8 mol/l) for 48 h, followed by western blot, and the cytoplasmic and nucleus of β-catenin was observed. (F) Effects of E 2 on the non-phosphorylated β-catenin (active β-catenin) in HESCs. Cells were stimulated with DMSO or E 2 (10 −8 mol/l) for 48 h, followed by western blot, and non-phosphorylated β-catenin was observed. (G) E 2 increases TCF transcriptional activity. TOPflash or FOPflash was co-transfected with pRL-SV40 into HESCs. After transfection for 24 h, the cells were stimulated with E 2 (10 −8 mol/l) and E 2 plus ICI for 48 h. The luciferase activities are shown as percentages of the control level. Data are expressed as mean± s.e.m . * P <0.05 and ** P <0.01 vs controls. Data presented are from three independent experiments.

Article Snippet: TOPflash, a TCF reporter plasmid; FOPflash, a negative control for TOPflash; and pRL-SV40, a Renilla luciferase expression plasmid, were purchased from GeneChem (Shanghai, China).

Techniques: Expressing, Cell Culture, Incubation, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Activity Assay, Transfection, 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