Journal: ACS nano

Article Title: Fracture-Targeted Delivery of β -Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing

doi: 10.1021/acsnano.7b05103

Figure Lengend Snippet: NP-delivered GSK-3β inhibitor increases β-catenin level and exhibited pH-responsive release, which enhances intracellular release of drug in the endolysosomal compartment. (A) NPs, TBP-NPs, and SCP-NPs were labeled with Texas Red fluorophore and exhibited robust cellular uptake. Cells were incubated with NPs at 0.1 mg/mL for 1, 4, and 24 h and stained with DAPI (blue) for cell nuclei. (B) Median fluorescent intensity quantified 1, 4, and 24 h after treatment via fow cytometry assay. N = 3, Mean ± SD. *p < 0.05 versus NP, all NP treated groups, and significantly different versus untreated control (2-way ANOVA and Tukey’s posthoc analysis). (C) Co-localization of FITC-labeled NPs (green) and lysosomes stained with Lysotracker (red). White triangles indicate areas of robust co-localization. (D) GSK-3β inhibitor release from PSMA-b-PS NPs. Loaded NPs were dialyzed against PBS at 37 °C with pH of 7.4 or 4.5. Mean ± SD, n = 3. (E) Active nuclear β-catenin level is represented by the ratio of TOPFlash to FOPFlash, normalized to cellular DNA concentration. Mean ± SD, n = 3, *p < 0.01 versus untreated control, #p < 0.0001 versus free drug (2-Way ANOVA with Tukey’s posthoc analysis).

Article Snippet: Cells were treated separately with 0.4 μ g/well TOPFlash and FOPFlash reporter plasmid (Addgene Plasmid #12456 and #12457 (Biechele and Moon, 2008)) using Lipofectamine LTX and PLUS reagents (Invitrogen) and high-glucose DMEM (without FBS or PSF) at 37 °C and 5% CO 2 for 3 h. 16 Transfection media was aspirated, and BME cell culture media (described in the previous section) with NP-loaded or free GSK-3 β inhibitor (10 μ M) was added to wells.

Techniques: Labeling, Incubation, Staining, Cytometry, Concentration Assay

Journal: The FASEB Journal

Article Title: The clock gene, brain and muscle Arnt-like 1, regulates adipogenesis via Wnt signaling pathway

doi: 10.1096/fj.12-205781

Figure Lengend Snippet: Inhibition of Wnt signaling pathway by attenuation of Bmal1 function. A, B) Gene expression analysis by quantitative RT-PCR in 10T1/2 cells (A) and Bmal1−/− MEF cells (B) at d 0–6; n = 3. HET, heterozygote; KO, knockout (homozygote). *P < 0.05, **P < 0.01; Student's t test. C, D) Immunoblot analysis of β-catenin protein level in Cyt fraction and total lysate in WT and Bmal1−/− MEF cells (C) and Bmal1-KD and Bmal1-overexpressing (cDNA) 10T1/2 cells with and without Wnt3a treatment (D). Right panels show quantification of cytosolic to total lysate ratio (n=3). *P < 0.05, **P < 0.01; ##P < 0.01. E) TOPFlash luciferase activity with and without Wnt3a in Bmal1-KD, Bmal1-overexpressing, and control 10T1/2 cells. Values are expressed as TOPFlash reporter activity normalized to Renilla readings after FOPFlash subtraction (n=4). **P < 0.01; ##P < 0.01.

Article Snippet: Per well, the transfection mixture contained 150 ng of Super TOPFlash (8xTCF binding sites) or FOPFlash (with mutated-TCF binding sites) luciferase reporter (Addgene 12456 and 12457; Addgene, Cambridge, MA, USA) as described by Randall Moon and colleagues ( 36 ), together with 20 ng of Renilla luciferase (pRL-TK, Promega, Madison, WI, USA) as an internal control.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Knock-Out, Western Blot, Luciferase, Activity Assay