Journal: eLife

Article Title: Pharmacological hallmarks of allostery at the M4 muscarinic receptor elucidated through structure and dynamics

doi: 10.7554/eLife.83477

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , AlphaScreen SureFire pERK 1/2 (Thr202/Tyr204) Assay Kits , PerkinElmer , , .

Techniques: Purification, Expressing, Plasmid Preparation, Recombinant, Dominant Negative Mutation, Amplified Luminescent Proximity Homogenous Assay, Software

Journal: BMC Cancer

Article Title: SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation

doi: 10.1186/1471-2407-14-987

Figure Lengend Snippet: Effect of SC-III3 on DNA damage and ATM/ATR pathways. (A) HepG2 cells were treated with SC-III3 for 24 hours. Western blot analysis of p-H2AX, total H2AX, p-Chk1 (Ser280) and p-Chk1 (Ser296) response to SC-III3. (B) HepG2 cells were treated with SC-III3 for 24 hours. Western blot analyses of the ATM/ATR signaling related proteins in HepG2 cells. (C) HepG2 cells were treated with 1 μM SC-III3 for different times as indicated. Western blot analyses of p-ATM, p-ATR, p-Chk1, p-Chk2, Cdc25C, p-CDK2, p53, and p21 in HepG2 cells. These experiments were done in triplicates.

Article Snippet: Antibodies against p-ATM (Ser1981), ATM, p-ATR (Ser428), ATR, p-Chk1 (Ser345), p-Chk1 (Ser280), p-Chk1 (Ser296), Chk1, p-Cdk2 (Tyr15), p53, p21 were purchased from Cell Signaling Technology (Danvers, MA). p-Chk2 (Thr68), Chk2, Cdc25A, p-H2AX(Ser139), H2AX antibodies were purchased from EnoGene Biotech (Nanjing, China), GAPDH monoclonal antibodies were purchased from Kangchen Bio-tech (Shanghai, China); Cdk2, cyclinA, cyclinE, cyclinB, Bax, Bcl-2 monoclonal antibodies were purchased from Bioworld (Georgia, USA).

Techniques: Western Blot

Journal: BMC Cancer

Article Title: SC-III3, a novel scopoletin derivative, induces cytotoxicity in hepatocellular cancer cells through oxidative DNA damage and ataxia telangiectasia-mutated nuclear protein kinase activation

doi: 10.1186/1471-2407-14-987

Figure Lengend Snippet: Role of ATM/Chk1/Chk2 in SC-III3-mediated S cell cycle arrest. (A, B) HepG2 cells were exposed to 1 μM SC-III3 for 24 h with or without adding of 10 μM Ku55933 or 0.3 μM UCN-01. Then, cells were analyzed for cell cycle distribution by flow cytometry. Cells in G0/G1, S, and G2/M phases were quantified and presented. (C) HepG2 cells were exposed to 1 μM SC-III3 with or without adding of 10 μM Ku55933 for 24 h. Then, western blot analyses of p-ATM, p-ATR, p-Chk1, p-Chk2, Cdc25C, p-CDK2, p53, and p21 in HepG2 cells. (D) HepG2 cells were exposed to 1 μM SC-III3 with or without adding of 0.3 μM UCN-01 for 24 h. Then, western blot analyses of p-Chk1, p-Chk2, p-Cdk2, and Cdc25A in HepG2 cells. The Data were presented as mean ± SEM for three separate experiments. The difference were significant at ** p < 0.01 compared with SC-III3 (1 μM) and ## p < 0.01 compared with SC-III3 (0 μM).

Article Snippet: Antibodies against p-ATM (Ser1981), ATM, p-ATR (Ser428), ATR, p-Chk1 (Ser345), p-Chk1 (Ser280), p-Chk1 (Ser296), Chk1, p-Cdk2 (Tyr15), p53, p21 were purchased from Cell Signaling Technology (Danvers, MA). p-Chk2 (Thr68), Chk2, Cdc25A, p-H2AX(Ser139), H2AX antibodies were purchased from EnoGene Biotech (Nanjing, China), GAPDH monoclonal antibodies were purchased from Kangchen Bio-tech (Shanghai, China); Cdk2, cyclinA, cyclinE, cyclinB, Bax, Bcl-2 monoclonal antibodies were purchased from Bioworld (Georgia, USA).

Techniques: Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Histone methyltransferase Smyd2 drives vascular aging by its enhancer-dependent activity

doi: 10.18632/aging.204449

Figure Lengend Snippet: Inhibition of Smyd2 alleviates Ang II-induced senescence associated phenotypes in vitro . ( A , B ) SA-β-gal staining ( A ) and EdU incorporation ( B ) assays in Ang II-induced RAECs with LLY-507 (3 μM) pretreatment. Representative staining images are shown in the figure left, and the statistical analysis of positive cells is shown in the figure right. Scale bars, 100 μm. ( C ) Expressions of senescence markers (p53, p21 and p16), pro-inflammatory molecules (VCAM-1 and COX-2) and DNA damage markers (p-Chk2 and γH2AX) were detected by western blot analysis upon LLY-507 pretreatment in Ang II-induced RAECs. Representative western blot images are shown on the left, and the statistical analysis of relative protein expressions is shown on the right. GAPDH was used as the loading control. ( D ) The mRNA levels of Nos2 (encoding the iNOS protein) in Ang II-induced RAECs with LLY-507 pretreatment was examined by RT-qPCR. ( E , F ) Immunofluorescence double staining of Smyd2 and the senescence markers (p53 and p16) upon LLY-507 pretreatment in Ang II-induced RAECs. Scale bars, 100 μm or 50 μm. Data are presented as the mean ± SEMs, * p < 0.05, ** p < 0.01, *** p < 0.001, each acquired from three individual experiments ( n = 3).

Article Snippet: Reagents and antibodies used in this study were obtained as follows: angiotension II (Ang II) was purchased from Meilunbio (Meilun Biotechnology, Dalian, China); the Smyd2 specific inhibitor LLY-507 was purchased from MedChem Express (MCE, USA); Smyd2, p53, p21, p16 and γ-H2AX antibodies were obtained from Cell Signaling Biotechnology (Danvers, MA, USA); H3K4me3, H3K4me1 and H3K27ac antibodies were purchased from Abcam (Cambridge, MA, USA); COX-2, VCAM-1 and GAPDH were purchased from Proteintech (Rosemont, IL, USA); p-ATM and p-Chk2 antibodies were purchased from Absci (MD, USA); IL-6 and iNOS antibodies were purchased from ABclonal (Wuhan, China).

Techniques: Inhibition, In Vitro, Staining, Western Blot, Quantitative RT-PCR, Immunofluorescence, Double Staining