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

Journal: Science Advances

Article Title: Microrobotic copper-rich electrochemical interfacing for targeted cancer theranostics in the gut

doi: 10.1126/sciadv.aeb5934

Figure Lengend Snippet: ( A ) Anticancer potency of Cu-CICs, formed by different duration and Φ, for HT-29 monolayer cells ( n = 18). ( B ) In vitro immunofluorescence imaging of key biomarker proteins (DLAT, FDX1, LIAS, and SDHB) involved in the cuproptosis process. ( C ) Viability of various GI cancer cell lines, including HT-29, NCI-N87 (human gastric cancer), HuTu80 (human duodenal cancer), PANC-1 (human pancreatic cancer), HCT116, LS174, and DLD1 (human colorectal cancers), after treatments with M-cuproptosis ( n ≥ 4). ( D ) Optical images and the corresponding live/dead assay results (top right) of HT-29 tumor spheroids with different sizes (0.3 mm for top and 1.3 mm for bottom) after treatment with 0, 1, 6, and 12 hour of M-cuproptosis. ( E ) In vitro adenosine triphosphate (ATP) assay results for HT-29 tumor spheroids (0.3, 1.3, and 2.5 mm) with increasing treatment duration ( n = 6 to 16). ( F ) Comparison of antitumor efficacy of 6-h M-cuproptosis fueled by Cu wires (diameter = 150 μm, P4-4 configuration) and the microrobot for 1.3-mm tumor spheroids ( n = 5 to 10). All data are means ± SD. All statistical analyses were performed using one-way analysis of variance (ANOVA). **** P < 0.0001. Scale bars, 50 μm (B) and 500 μm (D). A.U., arbitrary unit.

Article Snippet: Human GI cancer cell lines—including a colorectal adenocarcinoma cell lines HT-29 [American Type Culture Collection (ATCC)], LS174 (DSMZ), and DLD1 (DSMZ); a colorectal carcinoma cell line HCT116 (LGC Standards); a duodenal adenocarcinoma cell line HuTu 80 (ATCC); a pancreatic carcinoma cell line PANC-1 (DSMZ); a gastric adenocarcinoma cell line NCI-N87 (ATCC); an intestinal carcinoma cell line (Caco-2); and a colon epithelial cell line NCM460 (INCELL)—were purchased from each supplier and cultured according to adequate protocols.

Techniques: In Vitro, Immunofluorescence, Imaging, Biomarker Discovery, Live Dead Assay, ATP Assay, Comparison

Journal: PLoS ONE

Article Title: Downregulation of Genes Involved in Metabolism and Oxidative Stress in the Peripheral Leukocytes of Huntington's Disease Patients

doi: 10.1371/journal.pone.0046492

Figure Lengend Snippet: Summarized RT-PCR validation results of the 17 genes in human leukocytes.

Article Snippet: DLD , Hs01022655_m1 , ATTCTTGGACCAGGTGCTGGAGAAA.

Techniques: Biomarker Discovery, Binding Assay, Activation Assay

Journal: PLoS ONE

Article Title: Downregulation of Genes Involved in Metabolism and Oxidative Stress in the Peripheral Leukocytes of Huntington's Disease Patients

doi: 10.1371/journal.pone.0046492

Figure Lengend Snippet: Lists of assay ID and probe sequence for RT-PCR.

Article Snippet: DLD , Hs01022655_m1 , ATTCTTGGACCAGGTGCTGGAGAAA.

Techniques: Sequencing

Journal: Frontiers in Oncology

Article Title: ITGB1-mediated molecular landscape and cuproptosis phenotype induced the worse prognosis in diffuse gastric cancer

doi: 10.3389/fonc.2023.1115510

Figure Lengend Snippet: Association of ITGB1 with cuproptosis in DGC (A) Cuproptosis was stratified by ITGB1. (B) Correlations between ITGB1 and the cuproptosis-related-gene using Spearman analysis. The negative correlation was marked with blue and positive correlation with red. (C) Correlation analysis between ITGB1 and FDX1 in ACRG cohort. (D) Western blotting analysis of ITGB1, LIAS, DLD, DLST, DLAT, FDX1, PDHA1, PDHB and β-actin in MKN-45 cells with or without knockdown. (E) Western blotting analysis of ITGB1, LIAS, DLD, DLST, DLAT, FDX1, PDHA1, PDHB and β-actin in MKN-45 cells with or without overexpression. *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: The membranes were washed using 1% TBST by three cycles of 5 minute after incubation with Primary antibodies targeting ITGB1 (Proteintech, 12594-1-AP), FDX1 (Proteintech, 12592-1-AP), LIAS (Proteintech, 11577-1-AP), DLD (Proteintech, 16431-1-AP), DLST (abcam, ab177934), DLAT (Proteintech, 13426-1-AP), PDHA1 (Proteintech, 18068-1-AP), PDHB (Proteintech, 14744-1-AP), GLS (Proteintech, 29519-1-AP) and β-actin (Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Western Blot, Knockdown, Over Expression