Journal: Journal of Neuroinflammation

Article Title: Homer1 promotes the conversion of A1 astrocytes to A2 astrocytes and improves the recovery of transgenic mice after intracerebral hemorrhage

doi: 10.1186/s12974-022-02428-8

Figure Lengend Snippet: Homer1 protein and Selumetinib improve the pathological indexes and prognosis of ICH. A Effects of Homer1 protein and Selumetinib on cell activity [normal medium: F (3, 8) = 0.3731 P = 0.7748; ACM medium: F (3, 8) = 0.5067, P = 0.6885]. B Genotype identification of Homer1 flox/flox /Nestin-Cre +/− mice. C WB was used to detect the translation levels of C3 and S100A10 in the brain tissue of mice in each group on the 3rd day after ICH and the results were quantified in D [ F (4, 20) = 72.42, P < 0.0001] and E [ F (4, 20) = 183.6, P < 0.0001]. The blots are representative of other replicates in those groups. F expression of IL-1β [ F (4, 20) = 99.59, P < 0.0001]. G expression of TNF-α [ F (4, 20) = 98.31, P < 0.0001]. H Representative photographs of HE staining of brain tissue in each group. I Quantification of result in panel H [ F (4, 20) = 279, P < 0.0001]. J Representative photographs of Nissl staining of brain tissue in each group. K Quantification of result in panel J [ F (4, 20) = 196.6, P < 0.0001]. L Representative photographs of TUNEL staining of brain tissue in each group. M Quantification of result in panel L [ F (4, 20) = 159.6, P < 0.0001]. N Longa scores of different groups on the 3rd day after ICH [ F (4, 45) = 34.68, P < 0.0001]. O Survival curve of mice in each group ( n = 20) after ICH operation [Log-rank (Mantel–Cox) test: Chi-square = 21.4; df = 4; P = 0.0003]. The data were analyzed using one-way analysis of variance and all data are expressed as the mean ± standard deviation. * P < 0.05 represents a statistically significant difference between the two groups. ns no statistical difference

Article Snippet: The syringe was withdrawn after 10 min, and selumetinib (MCE, AZD6244) and Homer1 protein (EUPROTEIN, EP8767430) (5 mg/kg, dissolved in normal saline) were re-injected in situ for 10 min. After surgery, the skull hole was sealed with bone wax and the incision was closed with sutures.

Techniques: Activity Assay, Expressing, Staining, TUNEL Assay, Standard Deviation

Journal: International forum of allergy & rhinology

Article Title: Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma

doi: 10.1002/alr.21987

Figure Lengend Snippet: FGFR inhibition mitigates JNA fibroblast proliferation and MAPK activation. (A) JNA fibroblast lines were treated in triplicate with the indicated concentrations of AZD4547 for 72 hours and assessed for viability. Experiments were repeated 3 times and cumulative data were used to generate dose-response curves and determine the IC50 values for each line. (B) Levels of total and phosphorylated p44/42 MAPK were assessed by immunoblotting. (C) Cumulative densitometric values from 3 independent experiments of phosphorylated and total levels were graphed. FGFR = fibroblast growth factor receptor; IC50 = the dose that is cytotoxic to 50% of the cells treated in vitro; JNA = juvenile nasopharyngeal angiofibroma; MAPK = mitogen-activated protein kinase.

Article Snippet: Pan FGFR inhibitor AZD4547 was obtained from Chemietek (Indianapolis, IN), and VEGFR inhibitor SU5416 was obtained from Selleckchem (Houston, TX).

Techniques: Inhibition, Activation Assay, Western Blot, In Vitro

Journal: International forum of allergy & rhinology

Article Title: Inhibition of fibroblast growth factor receptor with AZD4547 mitigates juvenile nasopharyngeal angiofibroma

doi: 10.1002/alr.21987

Figure Lengend Snippet: FGFR inhibition mitigates migration and invasion of JNA fibroblasts. JNA fibroblast (A) migration and (B) invasion was measured in vitro in the presence of IC50 values of AZD4547 or DMSO. The number of migrating cells were normalized to the cell viability and represented graphically. All experiments performed in duplicate with 3 experimental repeats. DMSO = dimethylsulfoxide; FGFR = fibroblast growth factor receptor; IC50 = the dose that is cytotoxic to 50% of the cells treated in vitro; JNA = juvenile nasopharyngeal angiofibroma.

Article Snippet: Pan FGFR inhibitor AZD4547 was obtained from Chemietek (Indianapolis, IN), and VEGFR inhibitor SU5416 was obtained from Selleckchem (Houston, TX).

Techniques: Inhibition, Migration, In Vitro

Journal: Cell reports

Article Title: Microenvironmental control of hematopoietic stem cell fate via CXCL8 and protein kinase C

doi: 10.1016/j.celrep.2023.112528

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: AZD6244 , Cayman Chemical , Cat# 11599.

Techniques: Recombinant, Gene Expression, Sequencing, Modification, shRNA, Plasmid Preparation, Software, RNA Sequencing, Fluorescence, Microscopy, Live Cell Imaging

Journal: Nature Communications

Article Title: ERK-mediated NELF-A phosphorylation promotes transcription elongation of immediate-early genes by releasing promoter-proximal pausing of RNA polymerase II

doi: 10.1038/s41467-022-35230-4

Figure Lengend Snippet: a Sequence alignments of various NELF-A orthologues. Conserved residues are shown in blue. Green boxes, ERK phosphorylation sites (SP and TP). The Ser/Thr residues in which Ala substitution mutations were made (S363A, 4A, and 7A) are indicated. b HEK293 cells were cotransfected with His-NELF-A (WT, S363A, or 4A) and Myc-MEK1(DD), and treated with (+) or without (−) the MEK inhibitor (trametinib). Phosphorylation of His-NELF-A was analyzed by Phos-tag SDS-PAGE and immunoblotting using an anti-His antibody (upper). The expression level of Myc-MEK1(DD) in the cell lysates is also shown (lower). c HEK293 cells expressing Myc-NELF-A or its mutant derivatives (S363A, 4A, 7A, or 16A) were pretreated with or without a MEK inhibitor (AZD6244, 10 µM) and stimulated with TPA (300 nM, for 15 min). The phosphorylation status of immunoprecipitated Myc-NELF-A was assessed by ProQ diamond gel staining (top), and total Myc-NELF-A was subsequently monitored by SYPRO Ruby staining (second). d The reporter yeast cells expressing ERK(PD) and LexA-WW were transformed with ACT-NELF-A, ACT-NELF-A(4A), or the empty vector (ACT-alone), and were then spotted on selective plates with (+) or without (−) histidine. e HEK293 cells expressing Flag-NELF-A, -B, -C, or -E were stimulated with TPA for 15 min. Phosphorylation states of immunoprecipitated Flag-NELF subunits were detected by Pro-Q diamond gel staining (top), and their total protein levels were monitored by silver staining (second). The cell lysates were also separated by Phos-tag SDS-PAGE (third) or by conventional SDS-PAGE (fourth), and were immunoblotted with an anti-Flag antibody for phosphorylation states and total levels of the Flag-NELF subunits, respectively. c , e Cell lysates were probed for phosphorylated ERK1/2 (P-ERK) and ERK1/2 by immunoblotting (lower rows). Source data are provided as a Source Data file.

Article Snippet: Before stimulation, cells were pre-treated with MEK or ERK inhibitors [10 μM trametinib (LC Laboratories), 10 μM AZD6244 (LC Laboratories), or 10 μM FR180204 (Cayman)] for 45 min, a Cdk9 inhibitor [100 μM DRB (Cayman)] for 3 h, or with phosphatase inhibitors [400 nM okadaic acid, 0.2 μM FK506 (Wako), 20 μM cytostatin, 20 μM rubratoxin A (Institute of Microbiol Chemistry), or 0.5 μM sanguinarine (Cayman)] for 60 min as indicated, unless otherwise noted.

Techniques: Sequencing, Phospho-proteomics, SDS Page, Western Blot, Expressing, Mutagenesis, Immunoprecipitation, Staining, Transformation Assay, Plasmid Preparation, Silver Staining

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: (A) Bioliuminescence imaging (BLI; ventral image) of mice following intracardiac injection of MDA-MB-231-Luc2 cells. Mice were treated with AZD6244 (25 mg/kg) or vehicle control for 7 consecutive days (beginning at day 14 post cell injection), resulted in a significant attenuation of bioluminescent signals as quantified in (B). Regions of interest (red circles) drawn over the knees (C) and quantified (D) indicated a 10-fold drop in bioluminescent signals at day 21 in response to AZD6244 treatement. Data represented as mean ± SEM (vehicle, N=12; AZD6244, N=6). Asterisks indicate statistical significance (*** p<0.001).

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Imaging, Injection, Control

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: Representative histological sections of tumor-bearing knees were stained by the trichrome method. Vehicle control mice displayed large solid MDA-MB-231-Luc2 tumors that had developed beneath the growth plate, while the tumors in mice treated with AZD6244 had undergone central cavitation. Right panel shows a higher maginification view of the indicated knee region. Scale bar = 100 μm right panel, 1.25 mm left panel.

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Staining, Control

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: A 3 day treatment with AZD6244 (25 mg/kg) was performed on mice with late-stage established metastases (once the total photon fluxes of knees reached an average of 10 7 photons/s). Bioluminescence imaging (A) showed increased BLI signal in vehicle controls, while the AZD6244-treated mice remained unchanged, as quantified in (B). At the experimental end point (day 4 post-treatment initiation), vehicle-treated controls displayed >10 fold (~1 log) increase in tumor bioluminescence compared to AZD6244 treated mice. Data represented as mean ± SEM (vehicle, N=6; AZD6244, N=5). Asterisks indicate statistical significance (** p<0.01).

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Imaging

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: Representative tri-chrome stained images of regions of a late stage MDA-MB-231-Luc2 tibial metastasis demonstrates uniform tumor growth in a vehicle control (upper left; magnified in upper middle panel). In contrast, a large region of cell death is seen at the lower pole of an established metastasis following 3 days of treatment with 25 mg/kg AZD6244 (bottom left; magnified in lower middle panel). TUNEL staining (right panels; arrowheads indicating brown stain with methyl green counterstain) demonstrated the presence of TUNEL-positive cells in the late stage AZD6244 treated metastasis (right lower image). Scale bar = 100 μm right panel; 200 μm middle panel; and 1.25 mm left panel.

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Staining, Control, TUNEL Assay

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: Apoptosis was measured by Annexin V-FITC/propidium iodide assay. MDA-MB-231-Luc2 cells were plated in 10% or in 0.1% serum and treated with either 2.5 μm AZD6244, 2.5 μm camptothecin (CPT), or no treatment for 48 hrs prior to imaging. CPT treated cells showed increased cell death and apoptosis in full serum conditions (A and B). In contrast, low serum resulted in increased cell death in both CPT and AZD6244 treated groups (C and D). (E) Western blotting of MDA-MB-231-Luc2 cells exposed to AZD6244 under low serum conditions resulted in a dramatic increase of cleaved caspase-3 protein levels, relative to 10% serum conditions (N=3). Data represented as the mean ± SEM (N=3). Asterisks indicate statistical significance (*** p<0.001).

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Imaging, Western Blot

Journal: Oncoscience

Article Title: Selumetinib produces a central core of apoptosis in breast cancer bone metastases in mice

doi:

Figure Lengend Snippet: Annexin V-FITC/PI FACS plots and cell culture images after 48 hours in either 10% serum; upper panels) or low serum (0.1%); lower panels), and treated with either 2.5 μm AZD6244, 2.5 μm Camptothecin (CPT), or no treatment. CPT-treated cells had increased cell death and apoptosis in both serum conditions as indicated by the Annexin-V/PI double-stained cells in the FACS plots and the cellular morphology. However, a significant increase in cell death (Annexin-V/PI double-stained cells) or rounded-up morphology in the AZD6244 treated cells was seen only under low serum conditions.

Article Snippet: AZD6244 was purchased from Axon Medchem (Netherlands), camptothecin and MTT were from Sigma, and cell culture reagents from Invitrogen.

Techniques: Cell Culture, Staining

Journal: Nature Communications

Article Title: HSF1 phosphorylation establishes an active chromatin state via the TRRAP–TIP60 complex and promotes tumorigenesis

doi: 10.1038/s41467-022-32034-4

Figure Lengend Snippet: HeLa cells were infected with adenovirus expressing shRNA for PLK1, CSNK1A1, NEK7 or SCR ( a ), or treated with BI6727 (PLK1 inhibitor), FR180204 (ERK1/2 inhibitor), AZD6244 (MEK1/2 inhibitor), or rapamycin (mTOR inhibitor) ( b ). These cells were then treated with heat shock. Cell extracts were subjected to HSF1 immunoprecipitation and immunoblotting using antibody for HSF1 phospho-S419 or HSF1. Blue arrows indicate the positions of HSF1-S419 phosphorylated bands. The intensity of the upper band was markedly enhanced during heat shock. c PLK1 interacts with HSF1 in the nucleus during heat shock. Cell extracts were prepared as described in Fig. b and subjected to immunoblotting. Cells, in which endogenous PLK1 was replaced with GFP, wild-type HA-hPLK1, or its kinase-dead mutant, were treated with heat shock. Nuclear extracts were prepared and complexes co-immunoprecipitated using anti-PLK1 ( d ) or anti-HSF1 ( e ) were subjected to immunoblotting. Cells, in which endogenous PLK1 was replaced with GFP, HA-hPLK1, or HA-hPLK1-K82R, were treated with heat shock. ChIP-qPCR of HSF1, TRRAP, TRIM33, and PLK1 ( f ), or H2BK120ub and histone H2B ( g ) was performed. Norminal p -values were determined by one-way ANOVA, followed by Tukey-Kramer test in f and g . Error bars indicate SEM ( n = 3) in f and g . Experiments were repeated two times for a – e .

Article Snippet: Cells were treated with heat shock at 42 °C for the indicated periods, 5 mM L-azetidine-2-carboxylic acid (AZC; Tokyo Chemical Industry) for 6 h, 10 or 20 μM MG132 (Sigma-Aldrich) for 6 h, or 50 μM sodium arsenite (As; Sigma-Aldrich) for 6 h. Cells were also pretreated for 3 h before heat shock with inhibitors of kinase activity, including 100 nM BI6727 (PLK1 inhibitor, Toronto Research Chemicals), 10 nM BI2536 (PLK inhibitor, Cayman Chemical), 1 μM FR180204 (ERK1/2 inhibitor, Tokyo Chemical Industry), 20 nM AZD6244 (MEK1/2 inhibitor, ChemScene), and 30 nM rapamycin (mTOR inhibitor, LC Laboratories).

Techniques: Infection, Expressing, shRNA, Immunoprecipitation, Western Blot, Mutagenesis

Journal: British Journal of Cancer

Article Title: Combined effect of ALK and MEK inhibitors in EML4–ALK-positive non-small-cell lung cancer cells

doi: 10.1038/bjc.2011.586

Figure Lengend Snippet: Effects of the combination of the MEK inhibitor AZD6244 with TAE684 on signal transduction and apoptosis in lung cancer cells positive for EML4–ALK. ( A ) H2228 cells were incubated in the absence or presence of TAE684 (30 n M ), AZD6244 (1 μ M ), or both agents for 48 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins. ( B ) H3122 or H2228 cells were incubated in the absence or presence of the indicated concentrations of TAE684, AZD6244, or both agents for 60 h, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry. Data are means±s.e. from three independent experiments. * P <0.05 for the indicated comparisons; NS, not significant. ( C ) Nude mice with tumour xenografts established by subcutaneous implantation of H2228 cells were treated for 26 days by daily oral gavage with vehicle (control), TAE684 (0.5 mg kg −1 ), AZD6244 (25 mg kg −1 ), or the combination of both drugs. Tumour volume was determined at the indicated times after the onset of treatment. Data are means±s.e. for six mice per group. * P <0.05 for the combination of TAE684 and AZD6244 vs either drug alone. ( D ) Lysates prepared from tumour xenografts at the completion of the experiment in ( C ) were subjected to immunoblot analysis with antibodies to the indicated proteins.

Article Snippet: TAE684, crizotinib, and AZD6244 were obtained from ShangHai Biochempartner (Shanghai, China).

Techniques: Transduction, Incubation, Western Blot, Staining, Flow Cytometry, Control

Journal: British Journal of Cancer

Article Title: Combined effect of ALK and MEK inhibitors in EML4–ALK-positive non-small-cell lung cancer cells

doi: 10.1038/bjc.2011.586

Figure Lengend Snippet: Effects of the combinations of the MEK inhibitor, AZD6244, with either STAT3 depletion or survivin depletion on signal transduction and apoptosis in lung cancer cells positive for EML4–ALK. ( A ) H2228 cells were transfected with non-specific (−) or STAT3 siRNAs and incubated with or without AZD6244 (1 μ M ) for 48 h, after which cell lysates were subjected to immunoblot analysis with antibodies to the indicated proteins (left). Alternatively, the cells were transfected and treated with AZD6244 for 60 h, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry (right). ( B ) H2228 cells were transfected with non-specific (–) or survivin siRNAs and incubated with or without AZD6244 (1 μ M ) for 48 h, after which cell lysates were prepared and subjected to immunoblot analysis with antibodies to the indicated proteins (left). Alternatively, the cells were transfected and treated with AZD6244 for 60 h, after which the proportion of apoptotic cells was determined by staining with annexin V and propidium iodide followed by flow cytometry (right). All quantitative data are means±s.e. from three independent experiments. * P <0.05 for the indicated comparisons.

Article Snippet: TAE684, crizotinib, and AZD6244 were obtained from ShangHai Biochempartner (Shanghai, China).

Techniques: Transduction, Transfection, Incubation, Western Blot, Staining, Flow Cytometry