Fig 2: Diagram of Dectin-1-mediated microglial pyroptosis after ICH. The level of Dectin-1 is upregulated following ICH injury. Dectin-1 initiates SYK phosphorylation and mobilizes the NLRP3/caspase-1 downstream pathway to generate inflammatory mediators. In addition, the activated caspase-1 induced by Dectin-1 initiation cleaves GSDMD to release an N-terminal fragment, which forms pores on microglia, resulting in the extracellular release of inflammatory factors such as IL-1ß and IL-18. Inhibiting Dectin-1 by laminarin alleviates inflammatory responses, resulting in easing of intracerebral hemorrhage.

Fig 3: Treatment of CRC cell lines with C-13, an original non-enzymatic inhibitor of Syk.(A) Cells were exposed for 2 hours to various concentrations of C-13 and the impact of C-13 on cellular phenotype evaluated 72 hours post-treatment by: cell proliferation, viability and apoptosis, and normalized against DMF (vehicle)-treated HCT-116, DLD-1 and DIFI CRC cells. Data are the mean ±SD (error bars) of at least three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; compared with control). (B-C) Cells pretreated for 2 hours with C-13 or vehicle (DMF) were serum starved, labelled with CellTracker dye, and migrated toward FCS in Fluoroblock chambers. (D-E) Cells pretreated with C-13 or vehicle (DMF) were serum starved, labelled with CellTracker dye and seeded on Matrigel-coated Transwell inserts of 8 µm pore size (BD Falcon). FCS was used as chemoattractant in the Transwell lower part. After 24 hours at 37°C, migration was observed and counted microscopically. Images were obtained and total number of cells per field of view was measured using ImageJ software. Representative microscopy images are shown in Fig 6C and 6E. Error bars represent the mean number of migrated cells in triplicate wells, representative of three independent experiments (*, P<0.05; **, P<0.01; ***, P<0.001; compared with control). (F) DLD-1 cells pre-treated with the indicated concentrations of compound C-13 or DMF (vehicle) for 2 hours, were incubated with the recombinant human EGF (50 ng/mL) or a combination of EGF (50 ng/mL) + Cetuximab (50 µg/mL) for 10 minutes. Top panel- Protein extracts from cell lysates were analyzed by western blot using the indicated antibodies. Bottom panel- Densitometric quantification of immunoblot analyses of phospho-Erk (+EGF) versus phospho-Erk (-EGF) over control and phospho-Akt (+EGF) versus phospho-Akt (-EGF) over control. G) Syk-positive CRC cell lines DLD-1, SW620 and HCT-116; and Syk-negative breast cancer cells lines MDAMB231 and BT549 were exposed to various concentrations of C-13 for 72 h; (H) HCT-116, DLD-1 and HT29 cell lines were treated in parallel with C-13 (vehicle DMF) and R406 (vehicle DMSO) for 72 h; and cell viability was measured using CellTiter-Glo Luminescent cell viability assay (Promega). The relative changes were calculated and used to generate the bar graphs. The error bars represent the variation observed in three biological and three technical replicate. (*, P<0.05; **, P<0.01; ***, P<0.001; compared with Syk-negative MDAMB231 cell line for panel G; compared with vehicle for panel H).

Fig 4: Entospletinib-induced distinct downstream protein modifications in pre-B-ALL NALM-6. Western Blot analyses revealed distinct changes in the SYK downstream proteins in pre-B-ALL NALM-6. (a) Condensed conclusion of protein modifications in all tested B-ALL cell lines. (b) Schematic illustration of the SYK protein structure, showing the amino acids used to examine the phosphorylation status. (c) Representative Western blot images of NALM-6 cells after 72 h entospletinib exposure. (d) Western blot quantification of key proteins after 24 h, 48 h and 72 h exposure (=3 independent Western blots). Data are presented as the mean ± SD. Statistical significance was calculated by one-way ANOVA followed by Dunnett’s test as a post-hoc analysis and displayed as * p < 0.033, ** p < 0.002, *** p < 0.001 versus the control group (n = 3).

Fig 5: Basal characterization of SYK and pSYK expression in B-ALL cell lines. Characterization of basal SYK protein expression in B-ALL cell lines. Highest SYK expression was observed in pre-B-ALL NALM-6, while pro-B-ALL SEM showed the highest pSYK expression. (a) SYK and pSYK expression of two phosphorylation sites (Tyr525/526 and Y352) under non-stimulating conditions in the B-ALL cell lines and the DLBCL cell line SU-DHL-4 as well as the T-cell lymphoma cell line SUP-T1 (positive and negative control, respectively). (b) Immunofluorescent staining of SYK in the B-ALL cell lines and control cell lines (composite image of the Alexa Fluor Plus 488 green-labeled SYK and blue DAPI-stained nucleus) by confocal microscopy. (c) Intracellular staining of SYK-FITC and pSYK(Y348)-PE by flow cytometry in unstimulated B-ALL cells showing SYK and pSYK expression based on percentage (%) and mean fluorescent intensity (MFI) (SUP-T1 cell line as the negative control and IgG-stimulated SU-DHL-4 as the positive control). The mean fluorescence intensity (MFI) of all cell lines is expressed in arbitrary units (AU). Data are presented as the mean ± SD. Statistical significance was calculated by t-test and displayed as * p < 0.033, ** p < 0.002, *** p < 0.001 (n = 3).