Journal: Blood

Article Title: An open-label phase 2 trial of entospletinib (GS-9973), a selective spleen tyrosine kinase inhibitor, in chronic lymphocytic leukemia

doi: 10.1182/blood-2014-08-595934

Figure Lengend Snippet: Entospletinib shows greater selectivity for Syk than for R406. The blue circle represents Syk. Kinase activity for entospletinib is illustrated on the left, and kinase activity for R406 is illustrated on the right. Entospletinib is Syk-selective (Syk Kd = 7.6 nM, with only 1 other kinase with a Kd < 100 nM). R406 is nonselective (Syk Kd = 15 nM, with 25 kinases with Kd < 15 nM and 54 additional kinases with Kd < 100 nM).

Article Snippet: 12 Dissociation constant ( K d ) determinations of the strongest hits from the broad panel KINOME scan (DiscoveRx Corporation, San Diego, CA) showed that aside from Syk itself, only 1 kinase, TNK1, had a K d < 100 nM for entospletinib, whereas 79 kinases had K d < 100 nM for R406 ( ).

Techniques: Activity Assay

Journal: Blood Cancer Journal

Article Title: Depletion of STAT5 blocks TEL–SYK-induced APMF-type leukemia with myelofibrosis and myelodysplasia in mice

doi: 10.1038/bcj.2014.53

Figure Lengend Snippet: TEL–SYK induces activation of STAT5 in vivo . ( a ) Intracellular phospho flow cytometry for p-STAT5/6, p-PLCγ2 and p-SLP76 of GFP+ myeloid spleen cells coming from control, SYK wt , ITK–SYK and TEL–SYK-transplanted Balb/c mice. Graphs show the mean fluorescence intensity (MFI) per mouse. TEL–SYK shows highest activation of STAT5 and STAT6 in vivo , whereas SYK and ITK–SYK show a strong activation of PLCγ2 and SLP76. * P <0.05; ** P <0.01; *** P <0.001, unpaired t-test. ( b ) Intracellular phospho-flow cytometry for phosphorylated STAT5/6, PLCγ2 and SLP76 of 32D TEL–SYK cells, KG-1 cells (AML cell line) and SET-2 cells (megakaryocytic leukemia cell line) treated with the SYK inhibitor R406 for 5 min, 30 min or 2 h. For p-STAT6, p-PLCγ2 and p-SLP76 only the 30 min time point is shown. ( c ) 32D cells with IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells were all treated with R406 for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the dimethylsulfoxide (DMSO) control+s.e.m from three independent experiments ( n =3). ( d ) Total viable cell numbers were assessed by counting the cells with a Neubauer counting chamber after treatment with SYK inhibitor over 48–72 h. Graphs are shown for the DMSO control, the 2 μ M and the 4 μ M R406 concentration for 32D cells+IL-3, 32D TEL–SYK cells without IL-3, KG-1 cells and SET-2 cells. ( e ) 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 were treated with 5 and 10 μ M pimozide for 48 h and apoptosis was assessed by AnnexinV/7-AAD staining. Graph shows the mean relative percentage of viable cells (AnnexinV-/7-AAD-) compared with the DMSO control+s.e.m from three independent experiments ( n =3). * P <0.05; ** P <0.01; *** P <0.001, paired t -test. ( f ) Growth curves of 32D BCR–ABL and 32D TEL–SYK cells grown without IL-3 and treated with DMSO, 5 and 10 μ M pimozide were performed over 48 h in three independent experiments ( n =3) by counting Trypan blue negative cells in a Neubauer chamber.

Article Snippet: For SYK inhibition, the SYK inhibitor R406 (Axon medchem, Genentech, San Francisco, CA, USA) was solved in dimethylsulfoxide and used at a final concentration of 2 and 4 μM.

Techniques: Activation Assay, In Vivo, Flow Cytometry, Fluorescence, Staining, Concentration Assay

Journal: Molecular Medicine Reports

Article Title: In vitro and in vivo study of the expression of the Syk/Ras/c-Fos pathway in chronic glomerulonephritis

doi: 10.3892/mmr.2018.9355

Figure Lengend Snippet: mRNA levels of (A) Syk, (B) Ras, (C) MEK1/2, (D) ERK1/2 and (E) c-Fos in HBZY-1 cells were determined by reverse transcription-quantitative polymerase chain reaction. Syk, Ras, MEK1/2, ERK1/2 and c-Fos mRNA levels significantly increased in the LPS model group. The inhibitor R406 inhibited the LPS-induced activation of the Syk/Ras/c-Fos signaling pathway. Data are presented as the mean ± standard deviation of at least three independent experiments. **P<0.01 vs. normal group; ## P<0.01 vs. LPS group. Syk, spleen associated tyrosine kinase; MEK, mitogen activated protein kinase kinase; ERK, extracellular signal regulated kinase; p-, phosphorylated; LPS, lipopolysaccharide.

Article Snippet: The Syk/Ras/c-Fos pathway inhibitor R406 (inhibitor of Syk) was purchased from AbMole BioScience, (Shanghai, China).

Techniques: Reverse Transcription, Real-time Polymerase Chain Reaction, Activation Assay, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: In vitro and in vivo study of the expression of the Syk/Ras/c-Fos pathway in chronic glomerulonephritis

doi: 10.3892/mmr.2018.9355

Figure Lengend Snippet: Protein levels of (A) p-Syk, (B) Ras, (C) p-MEK1/2, (D) p-ERK1/2 and (E) c-Fos in HBZY-1 cells were determined by western blotting. p-Syk, Ras, p-MEK1/2, p-ERK1/2 and c-Fos protein levels significantly increased in the LPS model group. The inhibitor R406 inhibited the LPS-induced activation of the Syk/Ras/c-Fos signaling pathway. Data are presented as the mean ± standard deviation of at least three independent experiments. **P<0.01 vs. normal group; ## P<0.01 vs. LPS group. Syk, spleen associated tyrosine kinase; MEK, mitogen activated protein kinase kinase; ERK, extracellular signal regulated kinase; p-, phosphorylated; LPS, lipopolysaccharide.

Article Snippet: The Syk/Ras/c-Fos pathway inhibitor R406 (inhibitor of Syk) was purchased from AbMole BioScience, (Shanghai, China).

Techniques: Western Blot, Activation Assay, Standard Deviation

Journal: Glia

Article Title: Novel potent liposome agonists of triggering receptor expressed on myeloid cells 2 phenocopy antibody treatment in cells

doi: 10.1002/glia.24252

Figure Lengend Snippet: Triggering receptor expressed on myeloid cells 2 (TREM2)‐activating liposomes enhance TREM2‐mediated E. coli phagocytosis in THP1 cells. (a) Phagocytosis assay of pHrodo‐labeled E.coli in TREM2 antibody or liposome pre‐conditioned THP1. Briefly, 72 h PMA differentiated THP1 were pre‐incubated 6 h with vehicle, AF1828 (10 μg/ml), liposomes (50 μg/ml), cytochalasin D (5 μM), or spleen tyrosine kinase (Syk) inhibitor R406 (5 μM). Then, 60 μg/ml of pHrodo‐labeled E.coli was added to cells and cell fluorescence was acquired over time using Incucyte S3 (four well replicates per condition, four images per well). Specific cell fluorescence (from phagocytosed pHrodo) was calculated using Incucyte analysis software and plotted for TREM2 antibody stimulating effect over culture medium condition (mean ± SEM ) and are representative for three experiments. In top panel are representative images at end point. (b) Test of different liposomes on E.coli phagocytosis activity in THP1 and Trem2‐KO THP1 cells. Liposomes with different phospholipids (indicated in graphs) were tested in presence or not of Syk inhibitor R406 (5 μM) and compared to AF1828. In THP1‐KO cells, only dioleoyl‐phosphocholine (DOPS) and dipalmitoyl‐phosphocholine (DPPC) liposomes were tested in comparison to AF1828. (c) Graph corresponding to E.coli phagocytosis activity of WT THP1 cells upon AF1828 and indicated liposome treatments. TREM2 KO THP1 were used according to the same procedure without R406 treatment. Phagocytosis endpoint analysis for each presented condition in WT, upper panel, and KO THP1, lower panel. Multiple t test was used as statistical comparison method.

Article Snippet: For experiment including Syk Inhibitor VI R406 (505,819, Merck), starvation medium was added of 5 μM of R406.

Techniques: Liposomes, Phagocytosis Assay, Labeling, Incubation, Fluorescence, Software, Activity Assay, Comparison