Journal: PLoS ONE

Article Title: Canonical WNT Signaling Inhibits Follicle Stimulating Hormone Mediated Steroidogenesis in Primary Cultures of Rat Granulosa Cells

doi: 10.1371/journal.pone.0086432

Figure Lengend Snippet: Specific induction of WNT signaling is demonstrated by regulation of the WNT target gene, Axin2, transcriptional regulation of CTNNB1/TCF luciferase reporter and the transcriptional co-factor CTNNB1. Primary rat granulosa cells were treated for 24 h with vehicle or increasing doses of recombinant WNT3A (1, 5, 50, or 500 ng/mL) in the presence or absence of FSH (100 ng/mL). (A) Graphical representation of Axin2 mRNA expression and (B) tabular representation of Axin 2 mRNA expression data analyzed by real-time PCR (n = 5). (C) CTNNB1/TCF-dependent (TOPflash) transcriptional activity in primary GC (n = 3) and a GC tumor cell line (inset, n = 3). Statistical significance is presented as the mean ± standard error of the mean with significance set at ( P <0.05). Results of WNT treatment are compared within experimental groups incubated without (grey bars) and with (black bars) FSH treatment. Means with the same letter do not differ significantly. (D) Representative Western blot and quantitative analysis (n = 3) of CTNNB1 protein demonstrates an increase in CTNNB1 abundance after treatment with WNT3A alone ( P <0.05) and co-treatment of WNT3A + FSH ( P <0.01) compared with controls.

Article Snippet: Briefly, cells were transfected with 200 ng/well of TOPflash TCF Reporter Plasmid (Upstate Cell Signaling, Lake Placid, NY).

Techniques: Luciferase, Recombinant, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Incubation, Western Blot

Journal: BMC Cancer

Article Title: Wnt modulates MCL1 to control cell survival in triple negative breast cancer

doi: 10.1186/1471-2407-14-124

Figure Lengend Snippet: Wnt5B initiated WNT/ β-catenin to govern mitochondrial biogenesis through MCL1. (A) Dual luciferase assay of TCF promoter activity. Twenty thousand cells were seeded into 12-well plates, and the cells were transduced with shCtl or shWNT5B lenti-particles and treated with vehicle control or 50 nM mWNT5B. The plasmid Topflash and Renilla luciferase were co-transfected into MDA-MB-231 cells at 24 h using lipofectamine 2000. The cells were harvested for the dual luciferase assay at 48 h. Bars represent the average luciferase activities normalized to internal control (n = 3). ** P < 0.01; ***p < 0.001. (B) IHC staining of Myc and MCL1 in tissue array samples. FFPE tissue array slides were stained with Myc or MCL1 antibody, and the staining was graded into three different levels. Nuclear staining of Myc and cytosolic staining of MCL1 were observed. (C) Western blot of proteins related to mitochondrial structure and function. MDA-MB-231/shWNT5B and control cells were collected for cell lysates. The protein expression of MCL-1, AIF and PGC-1α was evaluated. (D) Western blot of Myc-regulated genes. MCL1 protein was inhibited with Myc knockdown by Myc siRNA; mitochondrial structural protein TOM20 also decreased. (E) MCL1 induced proteins in MDA-MB-231/shWNT5B cells. TOM20 was upregulated by MCL1 independent of WNT5B expression status.

Article Snippet: Cells at 80% confluence were co-transfected with TCF-driven Topflash reporter plasmid (Millipore) (1 μg) and control Renilla luciferease (20 ng) using 2.5 μl of Lipofectamine 2000 (Invitrogen).

Techniques: Luciferase, Activity Assay, Transduction, Plasmid Preparation, Transfection, Immunohistochemistry, Staining, Western Blot, Expressing