Journal: Scientific Reports

Article Title: Early life stress induces long-term changes in limbic areas of a teleost fish: the role of catecholamine systems in stress coping

doi: 10.1038/s41598-018-23950-x

Figure Lengend Snippet: Adrenergic receptor (α 2A ) expression at basal and post-stress conditions. Effect of treatment (control vs . early life stress regime at first feeding stage; FF and at all fins stage; AF), conditions (basal vs . acute stress) and the interaction between treatment and conditions (if it was maintained in the model which was indicated by “lack of fit” analysis) on mean (±SEM) density of the adrenergic receptor α 2A in the dorsal (Dld) and ventral (Dlv) parts of the dorsolateral pallium ( I ), the dorsomedial pallium subdivision 3 (Dm3) and the ventral part of the ventral telencephalon (Vv; II ). Capital letters indicate a treatment effect, small letters indicate an interaction effect and * indicates an acute stress effect. ANOVA Statistics for the Dld Treatment, F (2,18) = 1.1, p = 0.35 and Conditions: F (2,18) = 1.63, p = 0.22. Dlv Treatment, F (2,18) = 0.06, p = 0.94 and Conditions: F (2,18) = 0.03, p = 0.86. Dm3 Treatment, F (5,18) = 8, p = 0.003, Conditions: F (5,18) = 5.1, p = 0.04 and Interaction: F (5,18) = 4.1, p = 0.04 and the Vv Treatment, F (2,18) = 26, p < 0.001 and Conditions: F (2,18) = 6.9, p = 0.02.

Article Snippet: Sections for both basal and post-stress samples were then incubated together with primary antibodies for β 2 adrenergic receptors (dilution 1:50; rabbit polyclonal antibody; Santa Cruz Biotechnology, H-20, sc-569) and α 2A adrenergic receptors (dilution 1:70; goat polyclonal antibody, Santa Cruz Biotechnology, C-19, sc-1478) in 0.7% blocking solution in PBS-T at 4 °C overnight.

Techniques: Expressing, Control

Journal: Cell Death & Disease

Article Title: Role of non-genomic androgen signalling in suppressing proliferation of fibroblasts and fibrosarcoma cells

doi: 10.1038/cddis.2014.497

Figure Lengend Snippet: The AR/FlnA complex induces p27 Ser10 phosphorylation, stimulates migration and halts DNA synthesis in NIH3T3 cells treated with 10 nM R1881. In ( a ), quiescent NIH3T3 cells were left untreated or treated for 5 min with 10 nM R1881, in the absence or presence of the indicated concentrations of S peptide (1 or 10 nM). The peptide was added 30 min before hormonal stimulation. Lysates were incubated with anti-AR antibody and proteins in immune complexes were blotted with antibodies against the indicated proteins. In ( b ), quiescent cells on coverslips were left untreated or treated for 18 h with 10 nM R1881 in the absence or presence of the indicated concentration of S peptide. After in vivo pulse, BrdU incorporation was analyzed as above. In ( c ), quiescent cells were left untreated or treated with 10 nM R1881 in the absence or presence of the indicated concentrations of S peptide. Cells were allowed to migrate in trans-well chambers and migrated cells were scored by fluorescent microscopy. Data were expressed as relative increase. In ( d ), quiescent NIH3T3 cells were left untreated or treated for 30 min with 10 nM R1881, in the absence or presence of 10 nM S peptide. Control cells were treated with 10 nM S peptide alone. Lysate proteins were immune-blotted with antibodies against the indicated proteins. In ( e and f ), growing cells were transfected with non-targeting (ctrl siRNA) or FlnA (FlnA siRNA) siRNA. Cells were co-transfected with negative control siRNA Alexa-Fluor 488 to help identification of transfected cells. In ( e ), the western blot of lysate proteins from cells transfected with non-targeting (ctrl siRNA) or targeting (FlnA siRNA) siRNA with anti-FlnA antibody is shown. The filter was re-probed with anti-tubulin antibody as the loading control (tub). In ( f ), cells transfected with ctrl siRNA or FlnA siRNA were made quiescent and then left unstimulated or stimulated for 18 h with 10 nM R1881. After in vivo pulse, BrdU incorporation was analyzed by IF and expressed as % of transfected cells. In ( g ), cells transfected with ctrl siRNA or FlnA siRNA were made quiescent and then left unstimulated or stimulated with 10 nM R1881. Migrated cells were scored and data expressed as relative increase. In ( b , c , f and g ), data are derived from at least 500 scored cells for each experiment. Means and S.E.M. are shown

Article Snippet: AR was revealed using rabbit polyclonal anti-AR antibodies (N-20 or C19; Santa Cruz).

Techniques: Phospho-proteomics, Migration, DNA Synthesis, Incubation, Concentration Assay, In Vivo, BrdU Incorporation Assay, Microscopy, Control, Transfection, Negative Control, Western Blot, Derivative Assay

Journal: Experimental Animals

Article Title: Relationship between morphological development and sex hormone receptor expression of mammary glands with age in male rats

doi: 10.1538/expanim.17-0134

Figure Lengend Snippet: Immunohistochemical examination of the mammary glands in male rats. In the mammary gland at 35 days old (a, c, e), androgen receptor (AR) expression was not found (a) while estrogen receptor (ER) expression was found (c, arrows). Progesterone receptor (PgR) expression was observed in a small number of cells. The inset of (e) shows a PgR positive cell (arrow). In the oxyphilic cells at 100 days old (b, d, f), the portion of AR expression was high while the portions of ER and PgR were low. (a, b) AR, (c, d) ER, (e, f) PgR. Scale Bars, 25 µ m.

Article Snippet: The sections were incubated overnight at 4°C with diluted primary antibodies: mouse monoclonal anti human androgen receptor (AR; Perseus Proteomics Inc., Tokyo, Japan, clone H7507, dilution 1:1,000), mouse monoclonal anti human estrogen receptor (ER; Dako, clone 1D5, dilution 1:50) and rabbit polyclonal anti human progesterone receptor (PgR; Santa Cruz, Tokyo, Japan, clone C-19, dilution 1:1,000).

Techniques: Immunohistochemical staining, Expressing

Journal: Cell Death and Differentiation

Article Title: Extracellular anti-angiogenic proteins augment an endosomal protein trafficking pathway to reach mitochondria and execute apoptosis in HUVECs

doi: 10.1038/s41418-018-0092-9

Figure Lengend Snippet: AS and ISM are trafficked to mitochondria and enhance LE–Mito interaction. a AS and ISM treatment enhanced the presence of Rab7 in isolated mitochondria shown by WB. The mitochondrial fraction of AS, ISM, and FN-treated HUVECs was isolated and immunoblotted for the LE marker (Rab7), EE marker (Rab5), lysosome marker (LAMP2B), autophargosome marker (LC3B), ER marker (PDI), plasma membrane marker (VE-cadherin), cytosol marker (GAPDH), mitochondrial marker (VDAC), and ISM and ISM’s cell surface receptor (GRP78). A longer exposure of Rab7 WB is also included to show the presence of this protein in Mito under natural condition. b, c AS and ISM but not FN increased the association between LE and Mito as shown by confocal imaging. HUVECs labeled by the LE marker (Rab7-GFP) and MitoTracker (Red) were treated with AS, ISM, and FN. Colocalization between LE and Mito was determined by Pearson’s correlation coefficient. * *P < 0.01, n = 10. Scale bar, 5 μm. d , e AS and ISM enhanced the transfer of PKH26/R18-labeled lipid from the PM to mitochondria. FN treatment serves as a control. HUVECs labeled by PKH26 and R18 lipid dye were treated with AS, ISM and FN. Mitochondrial fraction was isolated and fluorescent intensity of the lipid dyes measured. ** P < 0.01, n = 3. Error bars denote SD. f , g AS and ISM significantly boosted the dynamics of LE targeted to Mito area shown by FRAP. Obviously shortened T 1/2 for fluorescence recovery of bleached LE (green) in Mito (red) area were observed upon ISM and AS treatment. HUVECs labeled by the LE marker (Rab7-GFP) and MitoTracker (Red) were treated with AS/ISM/FN and subjected to FRAP analysis. ** P < 0.01, n = 10. Error bars denote SD. Scale bar, 5 μm

Article Snippet: Antibodies for WB, immunofluorescent (IF) staining and co-IP were from Santa Cruz Biotechnology (Santa Cruz, CA, USA) including GRP78 (A10), NHERF1 (H100, A7), SNAP25 (H1), Rab5 (S19), Rab7 (H50), VDAC (D16), LAMP2B (H4B4), TOM20 (F10), AAC (N19), PDI (A1), VE-cadherin (C19), GAPDH (6C5) and β-actin (C4).

Techniques: Isolation, Marker, Cell Surface Receptor Assay, Imaging, Labeling, Fluorescence