Fig 2: Inhibition of SHP-2 impairs AM-induced dephosphorylation for pVE-cadherinY731 in HUVECs in vitro. (A, C) HUVECs were pre-incubated for 3 h with increasing concentration of SHP-2 inhibitors NSC-87877 (A) or SHP099 (C) and then treated with AM (10-9 M) for 6 h, followed by immunoblotting for VE-cadherin and pVE-cadherinY731, as indicated on the left. Blotting for ß-actin was used to control equal loading. (B, D) Relative changes in the pVE-cadherinY731 levels were assessed by quantification of immunoblots from five independent experiments. The value of control cells was set to 100%. *p < 0.05; **p < 0.01. (E, F) HUVECs were transfected with control siRNA (Ctrl siRNA) or with siRNAs directed against SHP-2. 48h later, cells were treated with AM (10-9 M) for 3 h and cell lysates were immunoblotted for pVE-cadherinY731, VE-cadherin, and SHP-2, as indicated on the left. Blotting for ß-actin was used to control equal loading. *p < 0.05; **p < 0.01. Each experiment is representative of five (B, D), and four (E) independent experiments. Results are shown as means ± SD.

Fig 3: Effect of SHP2 overexpression and p38 inhibition on CXCL8 levels.SHP2WT- and SHP2CS-transfected astrocytes were treated with PTP inhibitor, Na3VO4; p38 inhibitor, SB203580, or negative control inhibitor, SB202474. CXCL8 levels measured by ELISA in cellular supernatants collected 24 h post-treatment with A) Na3VO4 B) SB203580 C) SB202474. ND denotes not detectable. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons or student’s t-test.

Fig 4: PD-1 as a signaling receptor activates NF?B transcription factor in BTICs.(A) Schematic diagram of site-directed mutagenesis in two signaling motifs (ITIM, Y223F; ITSM, Y248F) of human PD-1 gene. (B) Representative flow cytometry plots of PD-1 expression in BT073 transfected with OE vectors coding wild-type (WT), 223, 248, or 223/248 mutant (Mut) version of PD-1. (C and D) ATP proliferation assay of human BT048 and BT073 cells OE wild-type or mutant versions of PD-1 compared with OE control. ns, not significant. (E) Heatmap of differentially expressed genes between PDCD1–OE cells and OE control of three human BTIC lines. Experiment was conducted once. (F) Volcano plot shows all data points that meet the false discovery rate (FDR) cutoff of 5% and 1.5-fold change criteria. (G) Validation of mRNA expression of TNFRSF19 and IKBKB genes by RT-qPCR in another culture set of BT048 and BT073 cells. Fold changes were calculated relative to PD-1 expression in OE control and normalized to GAPDH expression. (H) The phosphorylation of IKKa/ß, SHP-2, and NF?B (p65) was detected in the cell lysates of four human BTIC lines by Western blot. Actin was used as a loading control. Luminescence ATP proliferation test of PDCD1-OE BT048 and BT073 cells compared to OE controls after 72 hours of treatment with (I) SHP-2 inhibitor RMC4550 (3 Mµ) or (J) IKKa/ß inhibitor BMS-345541 (1 Mµ). DMSO, dimethyl sulfoxide. (K) After PD-1 immunoprecipitation of cell lysates and SDS–polyacrylamide gel electrophoresis, immunoblot was performed with antibodies for p–SHP-2, SHP-2, p-IKKa/ß, IKKß, and PD-1. Means were compared by unpaired (two-tailed) t test when comparing two groups. For more than two groups, one-way ANOVA with Tukey’s post hoc was used: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Data are represented as means ± SEM. See also fig. S5.

Fig 5: Depletion of Gas6 induces activation of the PTPN11 and MTOR pathways. (A) Typical expression pattern of Gas6 downstream target genes in GV and MII oocytes. For the PCR, a single oocyte-equivalent mRNA was used as a template for the amplification of each gene. GV and MII oocytes were harvested after in vitro culture for 0 and 16 hours, respectively. Expression levels were calculated from CT values after normalization with H1foo. The asterisks represent statistical significance at p < 0.05. (B) The qPCR analysis of Gas6 downstream target genes in Gas6-silenced MII oocytes, including Ptpn11, Bnip3 and Mtor. Expression levels were calculated from CT values after normalization with H1foo. Control, GFP dsRNA-injected MII oocyte; Gas6 RNAi, Gas6 dsRNA-injected MII oocyte. The asterisks represent statistical significance at p < 0.05. (C) Western blot analysis of p-PTPN11, PTPN11, BNIP3, p-MTOR and MTOR in Gas6-depleted MII oocytes. After Gas6 RNAi, expression levels of p-PTPN11, PTPN11, p-MTOR and MTOR were increased, whereas the expression of BNIP3 was decreased. A protein lysate from 200 MII oocytes was loaded into each lane. a-TUBULIN was used as a loading control.