Fig 2: LRP1 modulates SHP-2-mediated migration in response to PDGF-B.LRP expressing (B41) and deficient (LRP−/−) fibroblasts were seeded onto 5 µm costar transwell filters at 2×104 cells per well either in the presence or absence of the SHP-2 inhibitor, NSC-87877. After 3 h incubation at 37°C, either buffer (control) or PDGFB (30 ng/ml) was applied to the bottom chamber and incubation continued for 4 hours at 37°C. After incubation, the topsides of filters were cleared of cells with a cotton swab, and cells remaining on the bottom of the filters were fixed and the nuclei were stained with DAPI overnight. Filters were mounted onto glass slides and nuclei were quantified. (*p = 0.02, **p<0.0001, Students t-test).

Fig 3: MB suppress PD-1 signaling through blocking SHP2 recruitment by PD-1 AImpact of MB on the interaction between PD-1 and PD-L1. Jurkat-PD-1 cells (parental Jurkat cells served as negative control) were incubated with human PD-L1-Fc fusion protein in the presence of MB at indicated concentration at 4? for 30 min. Cells were stained for PE-conjugated anti-hIgG-Fc antibody. Binding of PD-L1 on cells was determined by flow cytometry.BLuciferase complementation analysis showing the effect of PD-1 Y248F mutation on the interaction between PD-1 and SHP2.CImpact of MB on luciferase activity of various engineered Jurkat T cells. JP-luc-sgSHP2: JP-luc cells treated with lentivirus expressing sgSHP2/CAS9 simultaneously. SHP099: JP-luc cells treated with 10 µM SHP099. JP-luc: Jurkat cell harboring NFAT-luciferase transgene and overexpressing PD-1.DImpact of MB on cytotoxicity of sgSHP2 or OT-1 in the presence of 10 µM SHP099 against EG7 or EG7-L1. EG7-L1: EG7 overexpressing PD-L1.ECo-IP analysis of impact of MB on interaction between SHP2 and EGFR.Data information: Data are representative of three independent experiments and were analyzed by unpaired t-test. Error bars denote SEM. *P < 0.05; ***P < 0.001.

Fig 4: pPDGFRβ kinase domain and pLRP1-ICD compete for SHP-2 binding.(a,b) Increasing concentrations of GST:pLRP1-ICD were incubated with microtiter wells coated with SHP-2 in the presence of 0, 20, 60 and 180 nM pPDGFRβ KD (a) or sPDGFr (b). Bound GST:pLRP1-ICD was detected with anti-GST antibody. Curves in (a) show best fits to a single class of sites using non-linear regression analysis. The KD values at each concentration are significantly different (p = 0.0007, Students t test). (c) Percent binding, normalized to 0 nM pPDGFRβ, of GST:pLRP1-ICD (100 nM) binding to SHP-2 in the presence of 20, 60 and 180 nM pPDGFRβ KD. (*, p = 0.0024; **p<0.0001, Students t test).

Fig 5: MB suppress PD-1 signaling through blocking SHP2 recruitment by PD-1 AConfocal cell images of Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry. Jurkat cells co-expressing PD-1-EGFP and SHP2-mCherry were incubated with 10 µg/ml of human PD-L1 protein in the presence of 100 nM and 1 µM of MB for 2 min, fixed with 4% paraformaldehyde, and stained with DAPI. Colocalization signal was examined with confocal microscope (scale bar = 2 µm).BFluorescence complementation analysis of the effect of MB on the interaction between PD-1 and SHP2. Jurkat cells co-expressing PD1-C-Luc or PD1Y248F-C-Luc and SHP2-N-luc were seeded in a 96-well plate precoated with 10 µg/ml of aCD3/aCD28 with media supplemented with 10 µg/ml human PD-L1 protein in the presence of MB at indicated concentrations for 6 h followed by analysis of luciferase activity.CCo-IP analysis of impact of MB on interaction between PD-1 and SHP2. 293T cells co-expressing PD-1 and SHP2-FLAG were treated with MB at indicated concentration for 6 h.DAbility of MB to block interaction between PD-1 with SHP2 revealed through ELISA. Y248-phosphorylated human PD-1 ITSM peptide was coated at 4 µg/ml in 96-well plate overnight. Recombinant human SHP2 protein incubated with the coated peptide in the presence of MB for 2 h. Bound SHP2 was measured by monitoring the activity of horse peroxidase conjugated on secondary antibody targeting SHP2.ERepresentative Western blot showing the levels of total and phosphorylated ZAP70, PKC?, and PLC?1 in the lysates of stimulated CTL, inhibited by PD-L1 in the presence of 1 µM of MB.FRepresentative Western blot showing the levels CD28 phosphorylation. CTL was stimulated with EG7 cells (parental), EG7-L1, and EG7-L1 in the presence of 100 nM of MB for 2 h. Protein immunoprecipitated (IP) with anti-pY from the lysates of the indicated CTL-EG7 co-culture was subjected to SDS–PAGE separation and blotted with aCD28. ß-Actin served as loading control. EG7-L1: EG7 overexpressing PD-L1.GPhos-tag analysis of CD28 phosphorylation in CTL. CTLs were stimulated with EG7 or EG7-L1 in the presence of 1 µM MB. Cell lysate was then separated by SDS–PAGE containing 50 µM of Phos-tag and blotted with aCD28 antibody. Phosphorylated CD28 (slow-moving) species were visualized through exposure.HFluorescence complementation analysis of the effect of MB on the interaction between CD28 with P85. Jurkat or Jurkat-PD-1 cells were co-transfected with CD28-C-Luc or CD28Y191F-C-Luc and P85-N-Luc. Cells were seeded in a 96-well plate coated with 10 µg/ml of aCD3/aCD28 in the presence of human 10 µg/ml of PD-L1 protein and 100 nM of MB for 6 h. Luciferase activity was measured as readout for CD28-P85 interaction.Data information: Data are representative of three independent experiments and were analyzed by unpaired t-test. Error bars denote SEM. *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available online for this figure.