Journal: British Journal of Cancer

Article Title: Loss of Coxsackie and adenovirus receptor downregulates α -catenin expression

doi: 10.1038/sj.bjc.6605331

Figure Lengend Snippet: Impact of CAR and α -catenin on cellular morphology. Cells after CAR knockdown appear round and smaller compared with controls (DLD1) or display less intercellular attachment sites (IEC-6). In matrigel, cells after CAR knockdown form amorphous clusters in contrast to the organised-appearing formations of matching controls. Ectopic ‘re’-expression of α -catenin partially reverses the effects seen after CAR knockdown when grown as a monolayer, and particularly results in organised cell formations similar to those of vector controls when cultured in matrigel. Images show representative results for DLD1 ( A ) and IEC-6 ( B ).

Article Snippet: The human colon cancer cell line DLD1 and the rat small intestine cell line IEC-6 were obtained from the American Type Culture Collection (Rockville, MD, USA) and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively, and were cultured in recommended growth media.

Techniques: Knockdown, Expressing, Plasmid Preparation, Cell Culture

Journal: British Journal of Cancer

Article Title: Loss of Coxsackie and adenovirus receptor downregulates α -catenin expression

doi: 10.1038/sj.bjc.6605331

Figure Lengend Snippet: Functional impact of CAR downregulation and α -catenin ‘re’-expression in DLD1 and IEC-6 cell lines. Proliferation ( A ), migration ( B ), and invasion ( C ) in DLD1 and IEC-6 were determined after CAR knockdown and ‘re’-expression of α -catenin in comparison with controls.

Article Snippet: The human colon cancer cell line DLD1 and the rat small intestine cell line IEC-6 were obtained from the American Type Culture Collection (Rockville, MD, USA) and from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), respectively, and were cultured in recommended growth media.

Techniques: Functional Assay, Expressing, Migration, Knockdown, Comparison

Journal: bioRxiv

Article Title: E4F1 COORDINATES PYRUVATE METABOLISM AND THE ACTIVITY OF THE ELONGATOR COMPLEX TO ENSURE PROTEIN TRANSLATION FIDELITY DURING NEURONAL DEVELOPMENT

doi: 10.1101/2022.12.19.521032

Figure Lengend Snippet: (A) RT-qPCR analysis of E4f1 mRNA levels and those of genes encoding key subunits or regulators of the pyruvate dehydrogenase complex (PDC) ( Dlat, Dld, Mpc1, Pdpr , and Pdha1 ) in the forebrain of E14.5 E4f1 (Nes)KO and CTL embryos (n=5 animals/group). (B) Immunoblot analysis of E4F1, DLAT, MPC1, DLD, PDHA1, and ACTIN (loading control) protein levels in the brain of E14.5 E4f1 (Nes)KO and CTL embryos. Right panel: Histobars represent the quantification of immunoblots performed on n=4 independent samples/group. (C) PDH activity in protein extracts prepared from E18.5 E4f1 (Nes)KO and CTL embryos (n=5 animals/group). (D) Blood lactate levels in E18.5 E4f1 (Nes)KO and CTL embryos (n≥40 animals/group). (E) IF analysis of MCT4 protein levels in brain sagittal sections prepared from E14.5 E4f1 (Nes)KO and CTL embryos. Fb= forebrain; Mb= middle brain; LV= lateral ventricule. Scale bars, 50 μm. Data are presented as mean + standard error of mean (SEM) for and or mean + standard deviation (SD) for and from the indicated number of animals. Statistical significance was evaluated using unpaired bilateral Student’s t -test (ns, not significant).

Article Snippet: Total protein extracts were prepared from isolated brains lysed in Laemmli buffer (80mM Tris pH=6,8, 2% SDS, 12% sucrose, 2% β-mercaptoethanol, bromophenol blue) and immunoblotting was performed using the following antibodies: anti-E4F1 (1/1000) , C-CASP3 (Cell Signaling, 9661S, 1/1000), DLAT (Santa Cruz, sc-271534, 1/500), MPC1 (Sigma, HPA045119, 1/1000), DLD (Santa Cruz, sc-365977, 1/1000), PDHE1 (Life Technologies, 459400, 1/500), ELP3 (Abcam, ab190907, 1/500), ELP1 (Abcam, ab62498, 1/500), TUBULIN (Sigma, T6199, 1/1000) and ACTIN (Sigma, A3854, 1/7000).

Techniques: Quantitative RT-PCR, Western Blot, Control, Activity Assay, Standard Deviation

Journal: Biomolecules

Article Title: The Proteomic Landscape of Parkin-Deficient and Parkin-Overexpressing Rat Nucleus Accumbens: An Insight into the Role of Parkin in Methamphetamine Use Disorder

doi: 10.3390/biom15070958

Figure Lengend Snippet: Parkin Deficit or Overexpression in Rat Nucleus Accumbens Changes Aerobic Respiration in Opposite Directions. ( A , B ) Gene set enrichment analysis (GSEA)-enrichment plots showing protein sets from GOBP_AEROBIC RESPIRATION signature significantly overrepresented ( p < 0.05) in parkin knockout (PKO) or parkin overexpression (PO) rat nucleus accumbens, as compared to wild-type nucleus accumbens. ( C , D ) Hierarchical clustering heatmap of the leading-edge proteins from the GOBP_AEROBIC_RESPIRATION ( p < 0.05) in PKO NAc and PO NAc. Three Krebs cycle enzymes, citrate synthase (CS), mitochondrial malonate dehydrogenase (MDH2), and dihydrolipoamide S-succinyltransferase (DLST), were decreased in PKO NAc and increased in PO NAc relative to wild-type NAc (red arrows).

Article Snippet: Proteins were transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA) at 400 mA, blocked with 5% non-fat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20), and incubated overnight at 4 °C with the following primary antibodies (diluted in 5% milk-containing TBST) against the following proteins: citrate synthase (CS) (NBP2-13878, 1:1000 Novus Biologicals, Centennial, CO, USA), dihydrolipoamide S-succinyltransferase (DLST) (PA5-51794, 1:1000,Invitrogen Life Technologies, Carlsbad, USA), mitochondrial malate dehydrogenase (MDH2) (9610, 1:1000, Cell Signaling, Danvers, MA, USA), dihydrolipoamide dehydrogenase (DLD) (NBP1-31302, 1:1000, Novus Biologicals, Centennial, CO, USA), 2-oxoglutarate dehydrogenase (OGDH) (15212-1-AP, 1:500, Proteintech Group Inc., Rosemont, IL, USA), mitochondrial complex I (mouse anti-NDUFS3 subunit, ab110246, 1: 2000, Abcam Inc., Waltham, MA, USA), complex II (mouse anti-SDHA subunit, ab14715, 1:2500, Abcam Inc., Waltham, MA, USA), complex III (mouse anti-UQCRC2 subunit, ab14745, 1:1000, Abcam Inc., Waltham, MA, USA), complex IV (rabbit anti-subunit IV, 4844, 1:1000, Cell Signaling Technology, Danvers, MA, USA), ß-actin (ab8227, 1:1000, Abcam Inc., Waltham, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2174, Cell Signaling Technology, 1:2000, Danvers, MA, USA).

Techniques: Over Expression, Knock-Out

Journal: Biomolecules

Article Title: The Proteomic Landscape of Parkin-Deficient and Parkin-Overexpressing Rat Nucleus Accumbens: An Insight into the Role of Parkin in Methamphetamine Use Disorder

doi: 10.3390/biom15070958

Figure Lengend Snippet: Parkin Deficit or Overexpression in Rat Nucleus Accumbens Changes Krebs Cycle Enzyme Levels in Opposite Directions. SDS-PAGE and western blotting showed statistically significant differences between parkin knockout (PKO) and parkin-overexpressing (PO) rats, as well as between these groups and wild-type (WT) controls. ( A ) dihydrolipoamide dehydrogenase (DLST), ( B ) mitochondrial malate dehydrogenase (MDH2), ( C ) 2-oxoglutarate dehydrogenase (OGDH), ( D ) dihydrolipoyl dehydrogenase (DLD), and ( E ) citrate synthase (CS). One-way ANOVA with Holm-Sidak’s post hoc test). * p < 0.05, ** p < 0.01, *** p < 0.001, n = 7/group. The data is expressed as mean ± SEM. ( F ) CS levels show a positive correlation with parkin levels ( p < 0.05, Pearson correlation test). Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: Proteins were transferred to PVDF membrane (EMD Millipore, Burlington, MA, USA) at 400 mA, blocked with 5% non-fat dried milk dissolved in TBST (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20), and incubated overnight at 4 °C with the following primary antibodies (diluted in 5% milk-containing TBST) against the following proteins: citrate synthase (CS) (NBP2-13878, 1:1000 Novus Biologicals, Centennial, CO, USA), dihydrolipoamide S-succinyltransferase (DLST) (PA5-51794, 1:1000,Invitrogen Life Technologies, Carlsbad, USA), mitochondrial malate dehydrogenase (MDH2) (9610, 1:1000, Cell Signaling, Danvers, MA, USA), dihydrolipoamide dehydrogenase (DLD) (NBP1-31302, 1:1000, Novus Biologicals, Centennial, CO, USA), 2-oxoglutarate dehydrogenase (OGDH) (15212-1-AP, 1:500, Proteintech Group Inc., Rosemont, IL, USA), mitochondrial complex I (mouse anti-NDUFS3 subunit, ab110246, 1: 2000, Abcam Inc., Waltham, MA, USA), complex II (mouse anti-SDHA subunit, ab14715, 1:2500, Abcam Inc., Waltham, MA, USA), complex III (mouse anti-UQCRC2 subunit, ab14745, 1:1000, Abcam Inc., Waltham, MA, USA), complex IV (rabbit anti-subunit IV, 4844, 1:1000, Cell Signaling Technology, Danvers, MA, USA), ß-actin (ab8227, 1:1000, Abcam Inc., Waltham, MA, USA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2174, Cell Signaling Technology, 1:2000, Danvers, MA, USA).

Techniques: Over Expression, SDS Page, Western Blot, Knock-Out