517 syk  (MedChemExpress)

Journal: Frontiers in Immunology

Article Title: Whole-Genome Expression Profiling in Skin Reveals SYK As a Key Regulator of Inflammation in Experimental Epidermolysis Bullosa Acquisita

doi: 10.3389/fimmu.2018.00249

Figure Lengend Snippet: Pharmacological inhibition of SYK blocks immune complex (IC)-induced activation of polymorphonuclear leukocytes (PMNs) in vitro and ex vivo . (A) Human PMNs were activated using IgG-ICs, and their activation was determined by measuring the release of reactive oxygen species (ROS). The data ( n = 11–30/group) were normalized to IC-activated PMNs in the presence of solvent (water) and are displayed as the median (black line), the 27/75 percentiles (box), and the 5/95 percentiles (error bars). BAY61-3606 reduced the release of ROS from IC-activated PMNs in a dose-dependent manner (ANOVA on Ranks with Dunn’s post-test). (B) Representative ROS release expressed as counts per second (CPS) over the 60-min experimental period. (C) Human PMNs were activated using IgA-ICs, and their activation was determined by measuring the release of ROS. The data ( n = 6/group) were normalized to the IC-activated PMNs in the presence of solvent (water) and are displayed as the median (black line), the 27/75 percentiles (box), and the 5/95 percentiles (error bars). BAY61-3606 reduced the release of ROS from IC-activated PMNs in a dose-dependent manner (ANOVA on Ranks with Dunn’s post-test). (D) Representative ROS release expressed as CPS over the 60 min experiment. (E) BAY61-3606 also ablates dermal–epidermal separation in cryosections of human skin incubated with anti-type VII collagen (COL7) IgG and PMNs. The data are presented as the mean (boxes) and STD (error bars) and are based on five experiments per group. To calculate whether the effects of BAY61-3606 were significant, ANOVA with Ranks and Dunn’s post-test was used. (F–H) Representative images of the cryosection assay at a 200× original magnification showing (F) no dermal–epidermal separation in the sections incubated with normal human serum (NHS) and PMNs, (G) no dermal–epidermal separation in the sections incubated with anti-COL7 IgG, and (H) no dermal–epidermal separation in the sections incubated with anti-COL7 IgG, PMNs, and 25 mg/ml BAY61-3606. (I–L) Representative experiments evaluating the expression of CD66b ( x -axis) and L-selectin (CD62L, y -axis) in immune complex-stimulated PMNs. (I) CD66b and CD62L expression in resting PMNs show low expression of CD66b and high expression of CD62L. Data are based on five experiments per group. (J) By contrast, IC activation leads to L-selectin shedding and increased CD66b expression. (K) Low concentrations of BAY61-3606 had no impact on the IC-induced changes in PMN surface molecule expression. (L) Higher compound concentrations normalized CD66b expression, but had no effect on L-selectin sheading. (M–P) These effects of BAY61-3606 were achieved at non-toxic concentrations, as evaluated by annexin V/propidium iodine staining. Representative results from (M) solvent- (water), (N) UV-irradiated- (positive control), and (O,P) BAY-61-3606-treated activated PMNs.

Article Snippet: Neutrophils (5 × 10 6 in 1 ml of RPMI 1640 containing 10% heat-inactivated FCS) were pre-incubated in presence or absence of 250 ng/ml of the Syk BAY 61-3606 (medchemexpress, Princeton, NJ, USA) inhibitor for 20 min at 37°C.

Techniques: Inhibition, Activation Assay, In Vitro, Ex Vivo, Solvent, Incubation, Cryosection Assay, Expressing, Staining, Irradiation, Positive Control

Journal: Frontiers in Immunology

Article Title: Whole-Genome Expression Profiling in Skin Reveals SYK As a Key Regulator of Inflammation in Experimental Epidermolysis Bullosa Acquisita

doi: 10.3389/fimmu.2018.00249

Figure Lengend Snippet: Pharmacological inhibition of SYK ablates signaling events in immune complex-activated polymorphonuclear leukocyte (PMN). (A) Representative blots from immune complex-activated PMN (“C”) or activated PMNs treated with BAY61-3606 (“BAY”) from three blood donors. (B) Image analysis showed that BAY61-3606 ablated pAkt phosphorylation and significantly reduced p38 and Erk phosphorylation. The data are presented as the mean (boxes) and STD (error bars) and are based on three experiments per group (* p < 0.05, t -test).

Article Snippet: Neutrophils (5 × 10 6 in 1 ml of RPMI 1640 containing 10% heat-inactivated FCS) were pre-incubated in presence or absence of 250 ng/ml of the Syk BAY 61-3606 (medchemexpress, Princeton, NJ, USA) inhibitor for 20 min at 37°C.

Techniques: Inhibition, Phospho-proteomics

Journal: Frontiers in Immunology

Article Title: Whole-Genome Expression Profiling in Skin Reveals SYK As a Key Regulator of Inflammation in Experimental Epidermolysis Bullosa Acquisita

doi: 10.3389/fimmu.2018.00249

Figure Lengend Snippet: Plaur and formyl peptide receptor 1 ( Fpr1 ) are regulated by SYK in immune complex-activated murine neutrophils. (A) Neutrophils were activated by immune complexes in absence or presence of BAY-61-3606 (BAY). Solvent and resting cells served as controls. Plots represent the expression of indicated gene in relation to Gapdh . Because of the non-parametric distribution, data are presented as median (centered vertical line), 25/75-percentile (boxes), and the 5/95-percentile (bars). Data are based on 8–10 samples per group. Statistical analysis was performed using ANOVA Ranks with the Student–Newman–Keuls post-test. (B) mRNA expression of Sykb, Gr-1, Plaur , and Fpr1 were determined in the skin of healthy mice (NR-IgG) and mice with experimental epidermolysis bullosa acquisita (EBA) (anti-type VII collagen IgG). For expression analysis, skin specimens from corresponding areas were obtained. Myeloid cell infiltration, mirrored by an increase in Gr-1 expression, was accompanied by an increased expression of Sykp, Plaur , and Fpr1 . Data are based on five mice per group. Statistical calculations were performed using Rank Sum test. (C) Western blot analysis of SYK and GAPDH expression in the same samples of immunization-induced EBA and normal mouse skin. SYK expression could, in most cases, be detected only in lesional skin—with very few SYK expression in healthy skin. The graph shows the relative amount of the mean gray value (MD) of the SYK bands per GAPDH bands. Here representative blots are shown. (D) Quantitative analysis of the Western blots from five mice per group ( t -test).

Article Snippet: Neutrophils (5 × 10 6 in 1 ml of RPMI 1640 containing 10% heat-inactivated FCS) were pre-incubated in presence or absence of 250 ng/ml of the Syk BAY 61-3606 (medchemexpress, Princeton, NJ, USA) inhibitor for 20 min at 37°C.

Techniques: Solvent, Expressing, Western Blot