Fig 2: STING deficiency down-regulates the activation of negative BCR signaling molecule, SHIP, and up-regulates the actin polymerization via enhancing the activation of WASP.Confocal analysis of splenic B cells from WT and KO mice labeled with AF546-mB-Fab-anti-Ig and then incubated without or with streptavidin for activation. Cells were fixed, permeabilized, and stained with Abs specific for pSHIP, pWASP, and F-actin (A and F). Splenic B cells from WT and Sting KO mice were activated with biotin-conjugated F(ab’)2 anti-mouse Ig(M + G) plus streptavidin for indicated times. Cell lysates were analyzed using SDS-PAGE and Western blot and probed for pSHIP/SHIP, pSHP1/SHP1 (C). Total protein or ß-actin was used as controls. Shown are representative images, blots, and the correlation coefficients between the labeled BCR and pSHIP (B), pWASP (G), as well as the relative intensity of bands from immunoblots (D and E) taken from three independent experiments. Splenic B cells were labeled with anti-B220 and stimulated with sAg for indicated times and then stained with phalloidin and antibody specific for pWASP for phos flow cytometry. MFI of pWASP and F-actin in B cells was quantified using FlowJo software (H and I). Shown are levels of pWASP and F-actin from one of three independent experiments. Scale bars, 2.5 µm. *P < 0.05; **P < 0.01.

Fig 3: TAM activated TLR4 signaling and inactivated immune checkpoint SHP1/SHP2. (A) The protein expression levels of the TLR4/NF-?B signaling pathway normalized to the GAPDH in RAW 264.7 cells were detected. (B) The expression levels of SIRPa protein, the phosphorylation levels of SHP1 and SHP2 proteins in RAW 264.7 cells were detected using Western blot. (C,D) The expression levels of SIRPa protein, the phosphorylation levels of SHP1 and SHP2 proteins in GH3 and AtT-20 cells were detected. (E) The expression levels of SIRPa protein, the activity of SHP1 and SHP2 proteins in tumor tissues were also detected. Bars indicates SEM, VC vehicle control, refers to tumor-bearing mice treated with the vehicle. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control or VC. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. cells treated with IL-TAM: tamoxifen.

Fig 4: SHP1-dependent ADP repression is SYK-dependent. NLCs were transfected with 50 nM scrambled siRNA control or three different SHP1-siRNAs (siRNA A–C). (A) Protein was harvested after 48 h and SHP1 and GAPDH expression visualized and quantitated by western blot to examine knockdown efficiency. (B) NLCs were co-cultured with autologous CLL cells for 48 h after SHP1 siRNA C or scrambled siRNA transfection. Cultures were then treated with or without obinutuzumab (10 µg/ml) for 2 h before ADP responses examined. Data presented as mean value ± SEM from five CLL patients. (C) Western blot data analysis of SYK (pSYKY525/526) and AKT phosphorylation (pAKTS473) in NLCs following SHP1 siRNA transfection. Representative blots and quantified data are shown. Data presented as mean value +/- SEM. Each point represents an individual patient. Statistical analysis used a Mann Whitney U Test. *p < 0.05, ***p, 0.001, and ****p < 0.0001.

Fig 5: SHP1 but not SHP2 is downstream of SIRPa. (A) Total and phosphorylated SHP1 and SHP2 expression were examined in phenotypically sensitive and resistant NLCs by western blotting. Representative blots and quantified data are shown. Each point represents a single patient. (B) NLCs were transfected with SIRPa siRNA C or scrambled control siRNA. SIRPa protein expression, as well as (C) total and phosphorylated SHP1 (pSHP1) or SHP2 (pSHP2) expression was estimated in transfected NLCs. Representative blots are shown and quantified data shown graphically. Data presented as mean value ± SEM and normalized to scrambled siRNA control. Each point represents an individual patient. Statistical differences were calculated by a Mann Whitney U Test. ***p < 001 and ****p < 0.0001. ns, not significant.