Journal: PLoS ONE

Article Title: Effect of Testosterone on Neuronal Morphology and Neuritic Growth of Fetal Lamb Hypothalamus-Preoptic Area and Cerebral Cortex in Primary Culture

doi: 10.1371/journal.pone.0129521

Figure Lengend Snippet: Shown is immunofluorescence labeling for androgen receptor (AR) and aromatase (Arom). Scale Bar = 50 μm.

Article Snippet: Immunocytochemistry was performed using the following primary antibodies: a mouse monoclonal anti-neuronal class III β-tubulin antibody (diluted 1:1000, Covance TUJ1, Covance Inc.; Princeton, NJ), PG-21 rabbit polyclonal anti-androgen receptor antibodies (1:100, Upstate 06–680; EMD Millipore; Billerica, MA) and rabbit polyclonal anti-aromatase antibodies (1:20, Acris Antibodies Inc, SanDiego, CA).

Techniques: Immunofluorescence, Labeling

Journal: International Journal of Molecular Sciences

Article Title: Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells

doi: 10.3390/ijms19072129

Figure Lengend Snippet: Loss of PTEN results in increased ROUTINE respiration in human and mouse cell lines ( A ) Schematic overview of the electron transfer through the mitochondrial complexes (CI–CII–CIV and CII–CIII–CIV) as part of the electron transfer system. NADH, the substrate for CI, is oxidized to NAD + (N-pathway). Succinate, the substrate for CII, is oxidized to fumarate leading to the reduction of FAD and formation of FADH 2 (S-pathway). ( B , C ) ROUTINE respiration expressed as O 2 flow per cell was determined by high-resolution respirometry in intact human and mouse cell lines. Androgen receptor (AR) and PTEN expression was validated by western blotting. Glycerinaldehyd-3-phosphate dehydrogenase (GAPDH) was used as internal loading control. Representative blots from three independent experiments are shown. ( D ) ROUTINE respiration expressed as O 2 flow per cell was determined in intact JP11066 Pten KO cells after 24 h treatment with 25 µM PI3K inhibitor LY29004 compared to mock control (DMSO). Data were expressed as mean and SEM of at least three independent experiments. Statistical differences were calculated with t -test and indicated with asterisks (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Article Snippet: The following primary antibodies were used: rabbit anti AR PG-21 (androgen receptor, dilution 1:500, Merck-Millipore, Burlington, CA, USA), rabbit anti PTEN (phosphatase and tensin homologe, 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), mouse anti GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase, dilution: 1:50,000, Merck-Millipore), and rabbit anti NaDC3 (dilution: 1:250, Proteintech).

Techniques: Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Succinate Accumulation Is Associated with a Shift of Mitochondrial Respiratory Control and HIF-1α Upregulation in PTEN Negative Prostate Cancer Cells

doi: 10.3390/ijms19072129

Figure Lengend Snippet: Effects of siRNA-mediated downregulation of NaDC3 on succinate-stimulated respiration. ( A ) Basal NaDC3 protein expression was evaluated in various PCa cell lines via western blotting. GAPDH was used as loading control. ( B ) LNCaP cells were transfected with 50 nM siNaDC3 and lipofectamine 2000. NaDC3 protein levels were determined and compared to a siCtrl and lipofectamine only (Mock) via western blotting. Numbers indicate relative ratios of densitometrical analysis of NaDC3 divided by the reference protein GAPDH. ( C ) Following treatment of LNCaP cells with siNaDC3 or the mock control, succinate-stimulated respiration was measured by high-resolution respirometry. To measure the effect on cell respiration by transporter-mediated uptake, succinate (S) was added to intact noncoupled (FCCP, carbonyl cyanide m-chloro phenyl hydrazine) cells and enhancement of respiration was calculated as S-pathway capacity in viable cells (vceS). Thereafter, digitonin was added to permeabilize the cells and determine stimulation of respiration by extracellular succinate bypassing cellular transporter systems (permeabilized cells, pce). Data were expressed as mean and SEM. Statistical differences were indicated in the graph (*, p < 0.05).

Article Snippet: The following primary antibodies were used: rabbit anti AR PG-21 (androgen receptor, dilution 1:500, Merck-Millipore, Burlington, CA, USA), rabbit anti PTEN (phosphatase and tensin homologe, 1:1000, Cell Signaling Technology Inc., Danvers, MA, USA), mouse anti GAPDH (glycerinaldehyd-3-phosphat-dehydrogenase, dilution: 1:50,000, Merck-Millipore), and rabbit anti NaDC3 (dilution: 1:250, Proteintech).

Techniques: Expressing, Western Blot, Control, Transfection