Journal: Molecular nutrition & food research

Article Title: Dietary wolfberry up-regulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

doi: 10.1002/mnfr.201200642

Figure Lengend Snippet: DNA copy numbers of cytochrome b (Cyt b) and β-actin were determined by real-time PCR using retinal genomic DNA and designed gene specific primers. Mitochondrial DNA copy number was expressed as Cyt b/β-actin (A). Mitochondrial mass in the retina was also determined by Western blot using antibody against Cox IV, a mitochondrial protein. The Cox IV protein level was normalized to β-actin in each sample (B). Retinal mitochondrial function integrity was tested by citrate synthase activity assay expressed as nmol/mg protein/min (C). Protein levels of TFAM in mitochondria were monitored by Western blot using anti-TFAM. Prohibitin was used as a loading control to normalize the TFAM values (D). Transcriptional and translational expression of transcription factors PGC-1α and NRF1 was analyzed by real time PCR (E, PGC-1α mRNA; G, NRF1 mRNA) and Western blot (F, PGC-1α protein; H, NRF1 protein). Data were normalized to β-actin. Representative Western blot images were shown. n=6. The statistical significance was at p<0.05 (*) and/or p<0.01 (**). mt DNA, mitochondrial DNA; Cyt b, cytochrome b; Cox IV, cytochrome c oxidase subunit IV; mt TFAM, mitochondrial transcription factor in mitochondria; NRF1, nuclear respiratory factor 1; PGC-1α, peroxisome proliferator-activated receptor γ coactivator-1α.

Article Snippet: Antibodies against GSTP1, BCO2, transcription factor A, mitochondrial (TFAM), and NRF1 were ordered from Proteintech (Chicago, IL).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Activity Assay, Control, Expressing

Journal: Molecular nutrition & food research

Article Title: Dietary wolfberry up-regulates carotenoid metabolic genes and enhances mitochondrial biogenesis in the retina of db/db diabetic mice

doi: 10.1002/mnfr.201200642

Figure Lengend Snippet: Hyperglycemia and/or subsequent hypoxia in db/db diabetic mice causes inhibition of lutein and zeaxnathin metabolic gene expression and decreases in protein levels of BCO2, AMPKα2, and TFAM in mitochondria, which leads to mitochondrial dysfunction and subsequent retinal degeneration in diabetes (marked with double lines). Dietary wolfberry and/or the bioactive components (wolfberry bioactives) primarily activates AMPKα2 in mitochondria and nuclei, which then triggers increased expression of genes related to lutein and zeaxanthin metabolism (SR-BI, GSTP1, and BCO2) and mitochondrial biogenesis (PGC-1α, NRF1, and TFAM), and decreases in cell stress responses (HIF-1α, VEGF, and HSP60), resulting in attenuation of hypoxia, enhancement of mitochondrial function, and subsequent retinal neuroprotection in db/db diabetic mice (marked with single lines).

Article Snippet: Antibodies against GSTP1, BCO2, transcription factor A, mitochondrial (TFAM), and NRF1 were ordered from Proteintech (Chicago, IL).

Techniques: Inhibition, Gene Expression, Expressing

Journal: Nature Communications

Article Title: The scaffold protein p140Cap limits ERBB2-mediated breast cancer progression interfering with Rac GTPase-controlled circuitries

doi: 10.1038/ncomms14797

Figure Lengend Snippet: ( a ) Protein extracts from two independent primary cancer cells for each genotype (NeuT-1, NeuT-2, p140-1 and p140-2) were run on 6% SDS–PAGE and stained with antibodies to NeuT, p140Cap and actin for loading control. ( b ) 10 6 cells as in a were injected in the left and right fat pads of nude mice. Tumour diameters were measured every week for 8 weeks. Two independent experiments were performed using five mice per group. Differences in tumour diameter were evaluated using two-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison post hoc tests (*** P <0.001). ( c ) Paraffin-embedded sections were prepared at the end of the experiments from tumours derived from mice as in b . Sections were analysed for Hematoxylin–Eosin (a,e), and for immunohistochemistry with antibodies to NeuT (b,f), PCNA (c,g) and activated Caspase3 (d,h). Representative images are shown. Scale bar, 50 μm. ( d , e ) Primary cancer cells for each genotype (NeuT-1, NeuT-2, p140-1, and p140-2) were plated in Matrigel/Collagen I 1:1 and left to grow for 15 days. Day 15 acina are shown as phase images in the left panels, or as Dapi nuclei staining (bright grey) in right panels. Arrows indicate the presence of invasive protrusions. Representative images from three independent experiments are shown. Scale bar, 50 μm. ( f ) The histogram represents the area of the acina quantified by the computer-generated software Zeiss Axiovision 4.5 and shown in arbitrary units (a.u.). ( g ) The histogram represents the percentage of acina structures with an internal lumen. The lumen has been manually quantified. In f and g , statistical significative differences were evaluated using unpaired t tests. Error bar: s.e.m. (*** P <0.001). ( h ) Primary cancer cells as in d were plated in Matrigel/Collagen I 1:1 and left to grow for 12 days. Protein extracts were run on 4–12% SDS–PAGE and stained with antibodies to Cleaved Caspase 3, Cyclin D1 and Actin for loading control. ( i ) Primary cancer cells as in d were analysed as day 15 acinar structures by immunostaining for a cis -Golgi matrix protein, GM130 (green), and a basal marker protein, beta1 integrin (red). Nuclei were co stained with DAPI (blue). Representative images are shown. Scale bar, 50 μm.

Article Snippet: 1:1,000) and anti phospho-Src (Tyr416; #2101, 1:1,000; Cell Signaling, Beverly, MA), anti N-cadherin (ab10203, 1:1,000) and anti GFP (ab13970, 1:500; Abcam, Cambridge, UK), anti c-ErbB2/c-Neu (Ab-3, OPL15, 1:1,000; Calbiochem, Merck KGaA, Darmstadt, Germany), anti Rac1 (#05–389 clone 23A8, 1:2,000), anti GAPDH (MAB374, 1:8,000) and anti p1248Y ERBB2 (#06–229, 1:1,000; Millipore, Billerica, MA, USA), anti beta1 Integrin CD29-PE (12-0299-41, 1:200; eBioscience, San Diego, CA, USA), anti GM130 (6,10,823, 1:300), anti p130Cas (6,10,272, 1:2,500) and E-Cadherin (6,10,182, 1:2,500; BD Transduction Laboratories, Franklin Lakes, NY), anti Tiam1 (C-16, 1:1,000), anti Actin (I-19, 1:1,000), anti Src (B-12, 1:1,000), and anti Cyclin D1 (H-295, 1:1,000; Santa Cruz Biotechnologies, Palo Alto, CA, USA), and anti Tubulin (T5168, 1:8,000; Sigma-Aldrich Co, Italy).

Techniques: SDS Page, Staining, Control, Injection, Comparison, Derivative Assay, Immunohistochemistry, Generated, Software, Immunostaining, Marker