syk inhibitor bay61–3606  (Cayman Chemical)

Journal: Scientific Reports

Article Title: BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts

doi: 10.1038/s41598-020-68628-5

Figure Lengend Snippet: Spleen tyrosine kinase (Syk) is activated by the incubation with 1% FBS or BAFF. ( A ) WiL2-NS cells were incubated with 20 ng/ml BAFF for 0.5, 1 and 2 h. ( B–D ) WiL2-NS cells were incubated in the RPMI 1,640 medium with 10% or 1% FBS in the presence or absence of BAY61-3606, Syk inhibitor ( C ) or BAFF ( D ). Cell lysates were prepared and western blotting was used to detect each phosphorylated Syk at tyrosine (Y)323, Y352 and Y525/526. All experiments were performed four times. Processing (such as changing brightness and contrast) is applied equally to controls across the entire image. Each band for Syk phosphorylation at Y525/526 was quantified by using ImageJ 1.34. Data in the bar graph represent the means ± SD. * p < 0.05; ** p < 0.01; significant difference as compared to BAFF-untreated control with 10% FBS. & p < 0.05; significant difference as compared to BAY61-3606-untreated control with 1% FBS ( C ). # p < 0.05; significant difference as compared to BAFF-untreated control with 1% FBS ( D ).

Article Snippet: BAY61-3606, Syk inhibitor, was purchased from Adooq bioscience (Irvine, CA, USA).

Techniques: Incubation, Western Blot

Journal: Scientific Reports

Article Title: BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts

doi: 10.1038/s41598-020-68628-5

Figure Lengend Snippet: Syk inhibitor enhanced MMP collapse by the incubation with 1% FBS. ( A , B ) WiL2-NS cells were incubated in the RPMI 1640 medium with 10% or 1% FBS in the presence or absence of BAY61-3606, Syk inhibitor and/or 20 ng/ml BAFF. Then, cells were stained with MitoProbe™ JC-1 reagent ( A ) or DiOC 6 ( B ) for the detection of MMP. Cells were observed and photographed with 400 × magnification under fluorescence microscope ( A , top). Number of high-red fluorescent cells was counted and represented as bar graph with mean ± SD ( A , bottom). Percentage of cells with low fluorescence of DiOC 6 was analyzed by flow cytometry ( B, top). Mean fluorescence intensity (MFI) in each histogram was analyzed with software FlowJo V10. MFI was represented as bar graph with the means ± SD ( B, bottom). All experiments were performed four times. ** p < 0.01; significant difference as compared to BAFF-untreated and BAY61-3,606-untreated control with 10% FBS. # p < 0.05; significant difference as compared to BAFF-untreated and BAY61-3,606-untreated control with 1% FBS. & p < 0.05; significant difference as compared to BAFF-treated and BAY61-3,606-untreated control with 1% FBS.

Article Snippet: BAY61-3606, Syk inhibitor, was purchased from Adooq bioscience (Irvine, CA, USA).

Techniques: Incubation, Staining, Fluorescence, Microscopy, Flow Cytometry, Software

Journal: Scientific Reports

Article Title: BAFF attenuates oxidative stress-induced cell death by the regulation of mitochondria membrane potential via Syk activation in WiL2-NS B lymphoblasts

doi: 10.1038/s41598-020-68628-5

Figure Lengend Snippet: Syk inhibitor increased B cell death by the incubation with 1% FBS. ( A–D ) WiL2-NS cells were incubated in the RPMI 1,640 medium with 10% or 1% FBS in the presence or absence of BAY61-3,606, Syk inhibitor and/or 20 ng/ml BAFF. Cell lysates were prepared and caspase 3 activity was measured by using Ac-DEVD-pNA, caspase 3 substrate. Caspase 3 activity was normalized with protein concentration ( A ). Total number of cells ( B ) or dead cells ( C ) were counted with hemocytometer and estimated by trypan blue staining, respectively. Cell lysates were prepared and western blotting was used to detect phosphorylated Syk at tyrosine (Y) 525/526. Processing (such as changing brightness and contrast) is applied equally to controls across the entire image ( D , left). Each band for Syk phosphorylation at Y525/526 was quantified by using ImageJ 1.34 ( D , right). All experiments were performed four times. Data in the line ( B ) or bar ( A-D ) graphs represent the means ± SD. * p < 0.05; ** p < 0.01; significant difference as compared to BAFF-untreated control with 10% FBS. # p < 0.05; significant difference as compared to BAFF-untreated and BAY61-3,606-untreated control with 1% FBS. & p < 0.05; significant difference as compared to BAFF-treated and BAY61-3606-untreated control with 1% FBS ( A , bottom). ( E ) This is the scheme for the regulation of serum deprivation (SD)-associated B cell survival via Syk-dependent mitochondria membrane potential (MMP). Our findings are indicated by gray-dotted lines. Black lines are from the results reported already in the literature.

Article Snippet: BAY61-3606, Syk inhibitor, was purchased from Adooq bioscience (Irvine, CA, USA).

Techniques: Incubation, Activity Assay, Protein Concentration, Staining, Western Blot