Journal: Journal of the American Heart Association

Article Title: Super‐Enhancer–Driven Syndecan‐4 Regulates Intercellular Communication in Hypoxic Pulmonary Hypertension

doi: 10.1161/jaha.124.036757

Figure Lengend Snippet: Figure 3. Silencing of syndecan-4 inhibited hypoxia-induced PASMC proliferation. A, Silencing of syndecan-4 blocked the effects of hypoxia on EdU incorporation and Ki67 expression in hPASMCs, EdU (red), Ki67 (green), 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 μm. B, hPASMCs viability was determined by Cell-Counting Kit-8 assay (n=6). C, Cell cycle analysis by flow cytometry indicated that hypoxia stimulated cell progression into G2/M+S phase, and this effect was inhibited by syndecan-4 small interfering RNA. D, Western blot analysis of syndecan-4, HIF-1α, PCNA, cyclin A, and cyclin D in hPASMCs (n=6). E, mPASMC viability was determined by Cell-Counting Kit-8 assay (n=6). F, Silencing of syndecan-4 blocked the effects of hypoxia on EdU incorporation in mPASMCs. EdU (red), 4′,6-diamidino-2-phenylindole (blue). Scale bar, 50 μm (n=6). G, Western blot analysis of syndecan-4, HIF-1α, PCNA, cyclin A, and cyclin D in mPASMCs (n=6). All values are presented as the mean±SEM. Statistical analysis was performed with 1-way ANOVA. *P<0.05, **P<0.01, ***P<0.001. EdU indicates ethynyl-2′- deoxyuridine; hPASMCs, human pulmonary artery smooth muscle cells; HYP, hypoxic; mPASMCs, mouse pulmonary artery smooth muscle cells; NC, negative control; NOR, normoxia; PASMC, pulmonary artery smooth muscle cell; PCNA, proliferating cell nuclear antigen; and SDC4, syndecan-4.

Article Snippet: Cells were incubated with anti- syndecan- 4 antibody (1:100, sc- 12766; Santa Cruz Biotechnology, Santa Cruz, CA), anti- PRKCA (1:100, 21991- 1- AP, Proteintech, Rosemont, IL), anti- CD63 antibody (1:100, ab315108, Abcam), and anti- Ki67 antibody (1:100, M00254- 3; Boster) at 4 °C overnight.

Techniques: Expressing, Cell Counting, Cell Cycle Assay, Flow Cytometry, Small Interfering RNA, Western Blot, Negative Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Testosterone-induced adult neurosphere growth is mediated by sexually-dimorphic aromatase expression

doi: 10.3389/fncel.2015.00253

Figure Lengend Snippet: Female and male NSPCs were grown as neurospheres without sex hormones for 4 days then allowed to adhere to a laminin substrate and expose to either 10 nM testosterone (T), 17β-estradiol (E 2 ), progesterone (P 4 ) for 20 min before Ki67/nestin double label fluorescent immunocytochemistry was performed (A, female control neurosphere shown). The proportion of DAPI/nestin-labeled cells expressing Ki67 per neurosphere was increased by 20 min of either T, E 2 or P 4 compared to controls in both females (B) and males (C) . Female and male neurospheres maintained without sex hormones were differentiated on a laminin substrate for 72 h ± 10 nM T, E 2 or P 4 when βIII-tubulin (red) immunocytochemistry (D) showed that while T, P 4 and E 2 increased the proportion of neuronal differentiation compared to controls, E 2 was more potent compared to T and P 4 in females (E) and males (F) . Results show mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.005 compared to control, # p < 0.05 compared to T and P 4 , one-way ANOVA with Tukey’s multiple comparisons test. Scale bar = 50 μM.

Article Snippet: Cells were then washed in ice-cold 0.1 M phosphate buffered saline (PBS) then fixed in 4% paraformaldehyde (PFA) on ice for 20 min. Immunocytochemistry was then performed; cells were blocked in 0.1 M PBS containing 5% normal donkey serum (Millipore) and 0.1% triton-X100 (Sigma-Aldrich) for 60 min at 20°C then incubated with primary antibodies, rabbit anti-Ki67 (1:300, DKSH Australia Pty Ltd., VIC, Australia), mouse anti-nestin (1:300, R and D Systems, MN, USA) for 3 h, washed in PBS then incubated in secondary antibodies, Alexa fluor 488-conjugated donkey anti-rabbit (1:400, Molecular Probes, Life Technologies) and Alexa fluor 555-conjugated donkey anti-mouse (1:400, Molecular Probes) then counterstained with 4′,6-Diamidino-2-Phenylindole (1:2000, DAPI, Sigma-Aldrich) for 10 min and coverslipped in Fluoromount (DAKO, North Sydney, NSW, Australia).

Techniques: Immunocytochemistry, Labeling, Expressing

Journal: Aging (Albany NY)

Article Title: Machine learning and single cell RNA sequencing analysis identifies regeneration-related hepatocytes and highlights a Birc5-related model for identifying cell proliferative ability

doi: 10.18632/aging.204775

Figure Lengend Snippet: Validation of liver regeneration-related signature post PHx. ( A ) A schematic of experimental design procedure: Mice underwent surgical resection. Ticks show the time point when mice were sacrificed. Liver tissues are harvested at 24h, 36h, 48h, 72h and 7days. ( B ) Time curve showed the liver-to-body weight of remanent liver after PHx. Data are mean±sd, ****p<0.0001, ns: no significant, (n=3 mice per groups). ( C ) The levels of serum indicate that liver mass return to normal function after 3days post PHx. ( D ) Relative expression levels of liver regeneration-related genes in hepatocytes between the PHx 0h group and the PHx 48h group. Data are “mean±sd”, ***p<0.001, ****p<0.0001. ( E ) Representative immunofluorescence shows multiplication capacity of hepatocytes at different time point post PHx, Ki67 (green), β-catenin (gray), DAPI (blue), Scale bar=100μm. Graphical representation indicates that the 36–48-hour time period represents an active mitotic phase. ( F ) We performed immunohistochemical imaging to evaluate the expression of Birc5 in both post-PHx and Sham livers. The significantly expressed hepatocytes were indicated by yellow arrows, scale bar=100μm. ( G ) Co-staining of zonal specific markers during homeostasis. Glul, which encodes glutamine synthetase (GS), was exclusively expressed in pericentral hepatocytes, while Cdh1, which encodes E-cadherin, was expressed only in periportal hepatocytes. ( H ) schematic of liver zonation. ( I ) Spatial distribution of Bircs5 following PHx at 36h and 48h revealed that liver regeneration-related hepatocytes primarily initiated in zone 2 before proceeding to other regions.

Article Snippet: The used antibody included: rabbit anti-GS (Abcam, 49873, 1:1000 dilution), mouse anti-CDH1(Biosciences, 610181, 1:50 dilution), rabbit anti-Survivin (Abconal, A1551, 1:50 dilution), rabbit anti-Ki67(Abbomax, 500-1874, 1:200 dilution), mouse anti-β-catenin (Biosciences, 610153, 1:400 dilution).

Techniques: Expressing, Immunofluorescence, Immunohistochemical staining, Imaging, Staining