Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: SHP2 is identified as a PLCγ-dependent VEGFR2 pY1175 interaction partner in endothelial cells (A) Representative western blots showing VEGFR2 immunoprecipitated with antibodies against RASA1, CSK, VAV2, or SHP2 in HUVECs unstimulated (−) or stimulated (+) with VEGFA (100 ng/mL, 5 min). Corresponding whole-cell lysates analyzed using antibodies against phosphorylated VEGFR2 (pY1175), total VEGFR2, and GAPDH as loading control. (B) Quantification of VEGFR2 interaction with RASA1, CSK, VAV2, and SHP2 from (A); n = 3 independent experiments. (C) Representative images of immunostainings for VEC (magenta), pVEC Y685 (green), and DAPI (blue) of unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pretreated with siCtr or siPTPN11. Scale bars: 30 μm. (D and E) Quantifications of MFI from (C), displayed as fold change relative to unstimulated control. (D) MFI of the VEC area. (E) MFI of pVEC Y685; n = 3 independent experiments, ≥3 fields of view per experiment. (F) Representative images of immunostainings for VEC (magenta), pVEC Y685 (green), and DAPI (blue) of unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pretreated with siCSK or siVAV2. Scale bars: 30 μm. (G and H) Quantifications of MFI from (F), shown as fold change over unstimulated control; n = 3 independent experiments, ≥3 fields of view per experiment. (I) Representative western blot showing VEGFR2 immunoprecipitated with antibodies against SHP2 or IgG control from unstimulated or VEGFA-stimulated (100 ng/mL, 5 min) HUVECs, pre-treated with siCtr or siPLCG1 . Corresponding whole-cell lysates analyzed by blotting with antibodies against pVEGFR2 Y1175, VEGFR2, PLCγ, and GAPDH as loading control. (J) Quantification of VEGFR2-SHP2 binding from (I); n = 3 independent experiments. (K) Representative western blot showing downstream VEGFA-induced signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs, for 2, 5, 10, and 20 min pretreated with siCtr or siPTPN11. (L–N) Quantification of western blots from (K), shown as fold change relative to unstimulated control. (L) Quantification of western blots for peNOS S1177. (M) Quantification of western blots for pSFK Y418. (N) Quantification of western blots for pVEGFR2 Y1175; n = 4–5 independent experiments. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. HUVECs, human umbilical vein endothelial cells; VEC, VE-cadherin; MFI, mean fluorescence intensity. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Western Blot, Immunoprecipitation, Control, Binding Assay, Fluorescence

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: Endothelial PLCγ/SHP2 signaling mediates activation of Src by regulation of both the activating and inhibitory tyrosine phosphorylation sites (A) PLA using antibodies against Src and pSFK Y418 to detect phosphorylation of Src on Y418 in unstimulated or VEGFA-stimulated HUVECs (100 ng/mL, 5min), pretreated with siCtr or siPTPN11 . Endothelial junctions are stained for VEC (magenta) and nuclei with DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at higher magnification in panels below. Scale bars: 10 μm. (B and C) MFI quantifications from (A), displayed as fold change relative to unstimulated control. (B) MFI of the total PLA signal. (C) MFI of the junctional PLA signals representing Y418 phosphorylation of Src; n = 7 independent experiments, ≥3 fields of view per experiment. (D) Representative western blot showing pSFK Y529 signaling in unstimulated (−) or VEGFA-stimulated HUVECs (100 ng/mL) for 2, 5, 10 and 20 min, pretreated with siCtr or si PTPN11 . (E) Quantification of western blots from (D); n = 5 independent experiments. (F and G) PLA using antibodies against Src and pSFK 529, visualizing phosphorylation of Src at the inhibitory phosphosite in HUVECs stimulated for 2, 5, and 10 min or left unstimulated. (F) Phosphorylation of Src at the inhibitory phosphosite in HUVECs pre-treated with siCtr . (G) Phosphorylation of Src at the inhibitory phosphosite in HUVECs pre-treated with siPTPN11 . Endothelial junctions are stained for VEC (magenta) and nuclei with DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at a higher magnification in panels below. Scale bars: 10 μm. (H) Quantification of PLA experiments from (F) and (G) and F; n = 6 independent experiments, ≥3 fields of view per experiment. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. PLA, proximity ligation assay; HUVECs, human umbilical vein endothelial cells; VEC, VE-cadherin. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Activation Assay, Phospho-proteomics, Staining, Control, Western Blot, Proximity Ligation Assay

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: PLCγ/SHP2 interplay leads to eNOS activation followed by Src nitration (A) Representative western blot showing eNOS T495 signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs for 2, 5, 10 and 20 min, pre-treated with siCtr or siPTPN11 . (B) Quantification of western blots from (A); n = 4 independent experiments. (C) PLA for NitroTyr and pSFK Y418 to detect full activation of Src in HUVECs stimulated with VEGFA (100 ng/mL, 5 min) or left unstimulated, and pretreated with siCtr or siPTPN11 . Endothelial junctions are stained for VEC (magenta) and DAPI (blue). Scale bars: 30 μm. Boxed regions in the upper panels are shown at higher magnification in panels below. Scale bars: 10 μm. (D) Quantification of junctional MFI PLA signals representing Y418 phosphorylation and 3-nitration of Src from (C), displayed as fold change to unstimulated control; n = 5 independent experiments, ≥3 fields of view per experiment. (E) Western blot showing eNOS S1177 and SHP2 Y542 signaling in unstimulated (−) or 100 ng/mL VEGFA-stimulated HUVECs for 2, 5, 10, and 20 min, pre-treated with siCtr or siPLCG1 . (F and G) Quantifications of western blots from (E). (F) Quantifications of western blots for peNOS S1177. (G) Quantifications of western blots for pSHP2 Y542; n = 4 independent experiments. (H) Representative immunostaining images with antibodies against VE-cadherin (VEC; magenta) and pSHP2 Y542 (green), in HUVECs unstimulated or stimulated with VEGFA (100 ng/mL, 5min) after downregulation with siCtr or siPLCG1 . Scale bars: 30 μm. (I) Quantification of MFI from (H), shown as fold change relative to unstimulated control; n = 4 independent experiments, ≥3 fields of view/experiment. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. VEC, VE-cadherin. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Activation Assay, Nitration, Western Blot, Staining, Phospho-proteomics, Control, Immunostaining

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: VEGFR2 pY1173/PLCγ-induced vascular permeability in vivo requires SHP2 (A) Representative immunostaining images with antibodies against VEC (magenta) and pSHP2 Y542 (green) in the back skin of WT and Plcg1 iECKO mice after intradermal injection of PBS or VEGFA. Scale bars: 50 μm. (B) Quantification of MFI values from (A), for vascular pSHP2 Y542, displayed as fold change relative to PBS control; n = 5 mice/genotype, ≥3 fields of view/mouse. (C) Representative images of immunostaining for VEC (magenta) and pVEC Y685 (green) in the back skin of WT mice, intradermally injected with DMSO or SHP099 and subsequently PBS or VEGFA at the same site. Scale bars: 50 μm. (D) Quantification of MFI values from (C), for vascular pVEC Y685, shown as fold change relative to PBS control; n = 5 (DMSO) and 4 (SHP099) WT mice, ≥3 fields of view/mouse. (E) The Miles assay showing Evans blue leakage in the back skin of DMSO (control) or SHP099 treated WT mice, intradermally injected with PBS or VEGFA. (F) Quantification of extravasated Evans blue from (E), shown as fold change of DMSO-PBS-treated mice; n ≥ 8 mice/condition. One-way ANOVA. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. VEC, VE-cadherin. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Permeability, In Vivo, Immunostaining, Injection, Control

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: VEGFR2 Y1173 heterozygosity is accompanied by decreased tumor endothelial PLCγ/SHP2 signaling and tumor vascular leakage (A) Representative immunostaining images of Vegfr2 +/+ (WT) and Vegfr2 Y1173F/+ (Y1173F/+) B16F10 melanoma tumors, showing vessels (IB4; red), pSHP2 Y542 (yellow), and fibrinogen (green). (B and C) Quantification of MFI from (A), shown as fold change over WT. (B) MFI for pSHP2 Y542. (C) MFI for fibrinogen; n = 5 (WT) and 3 (Y1173F/+) mice, ≥3 fields of view/mouse. (D) Representative immunostaining images of Vegfr2 +/+ (WT) and Vegfr2 Y1173F/+ (Y1173F/+) B16F10 melanoma tumors, showing vessels (CD31; red), pVEC Y731 (green), and CD45 (cyan). (E and F) Quantification of MFI from (D), displayed as fold change of WT. (E) MFI for pVEC Y731. (F) MFI for CD45 + cells (F); n = 5 (WT) and 3 (Y1173F/+), ≥3 fields of view/mouse. Scale bars: 100 μm. (G) Representative immunostaining images of WT and Plcg1 iECKO B16F10 melanoma tumors, showing vessels (IB4; red), pSHP2 Y542 (yellow), and fibrinogen (green). (H and I) MFI quantifications from (G), shown as fold change of WT. (H) MFI for pSHP2 Y542. (I) MFI for fibrinogen; n = 7 (WT) and 7 ( Plcg1 iECKO ), ≥3 fields of view/mouse. Unpaired 2-tailed Student’s t test. Data represent the mean ± SD. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. MFI, mean fluorescence intensity. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Immunostaining, Fluorescence

Journal: iScience

Article Title: SHP2 regulates VEGFR2 Y1175/PLCγ signaling to impair tumor endothelial barrier stability

doi: 10.1016/j.isci.2026.114784

Figure Lengend Snippet: SHP2 and PLCγ are clinically relevant biomarkers for tumor vascular leakage (A and B) Representative multiplex images of RCC patient biopsies. (A) Multiplex images for RCC patients with low vascular PLCγ and SHP2 expression. (B) Multiplex images for RCC patients with high vascular PLCγ and SHP2 expression. Tissue sections were stained for PLCγ (red), SHP2 (yellow), CD34 (green), and FpA (white) to visualize vascular leakage, and counterstained with Hoesht. Scale bars: 100 μm. Boxed regions are shown at higher magnification to the right. Scale bars: 30 μm. (C) Heatmap comparing endothelial SHP2 expression, PLCγ expression, and vascular leakage scores across individual RCC patients ( n = 16). Patients are arranged by their ID number, with color coding indicating high (green), medium (yellow), or low (red) levels. (D) Schematic for the mechanism of SHP2/PLCγ interaction upon VEGFR2 activation. In summary, VEGFA stimuli leads to the phosphorylation of VEGFR2 Y1175, which recruits both PLCγ and SHP2. SHP2 is needed for the activation of PLCγ at the plasma membrane and hydrolysis of PIP2 to IP3 and DAG. The former triggers the release of intracellular calcium (Ca 2+ ), and together with DAG, activates PKC, causing phosphorylation of the eNOS activating site S1177 and dephosphorylation of the inactivating site T495. Production of NO mediates nitration and full activation of Src, which phosphorylates Y685 of VEC, disrupting the adherens junctions and resulting in vascular leakage. Created in BioRender. Kremmyda, P. (2025). RCC, renal cell carcinoma; FpA, fibrinopeptide A; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; PKC, protein kinase C; eNOS, endothelial nitric oxide synthase; NO, nitric oxide; VEC, VE-cadherin. See also .

Article Snippet: SHP2 chemical inhibition was performed by treating cells with 30 μM SHP099 (MedChemExpress, HY-100388) in Dimethyl sulfoxide (DMSO) diluted in starvation medium for 3 h. Starvation media with DMSO was used as the unstimulated control.

Techniques: Multiplex Assay, Expressing, Staining, Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, De-Phosphorylation Assay, Nitration

Journal: Scientific Reports

Article Title: SHP2 promotes osteosarcoma via regulating STAT3/TET3/HOXB2 signaling

doi: 10.1038/s41598-026-35493-7

Figure Lengend Snippet: SHP2 overexpression promotes osteosarcoma proliferation by regulating the STAT3/TET3/HOXB2 and c-Myc/NANOG/NUSAP1 signaling pathways. ( A ) Western blot analysis of protein expression bands in 143B and MG63 cells, with statistical graphs showing relative protein expression levels. Groups: NC, SHP2-OE, SHP2-OE + Stattic, SHP2-OE + Stattic + TET3-OE, and SHP2-OE + Stattic + TET3-OE + HOXB2-KD. ( B ) Colony formation assay results assessing the proliferative capacity of 143B and MG63 cells, with statistical analysis graphs of colony numbers. ( C ) CCK-8 assay results showing the proliferative capacity of 143B and MG63 cells, with statistical analysis of proliferating cell counts. Data are presented as mean ± standard deviation. N = 3; P < 0.05 was considered statistically significant; ns P > 0.05, * P < 0.05, ** P < 0.01.

Article Snippet: Fetal bovine serum (FBS) was purchased from Global Kang Qinhuangdao Biotechnology Co., Ltd.; DMEM basal medium, penicillin-streptomycin solution (double antibiotics), phosphate-buffered saline (PBS), and 0.05% trypsin were obtained from Wuhan Procell Life Science & Technology Co., Ltd.; Matrigel was sourced from Corning, USA; Transwell chambers, dimethyl sulfoxide (DMSO), and SDS-PAGE protein loading buffer were acquired from Shanghai Beyotime Biotechnology Co., Ltd.; High-efficiency RIPA lysis buffer and protease phosphatase inhibitors were purchased from Beijing Solarbio Science & Technology Co., Ltd.; The BCA protein concentration assay kit was obtained from GLPBIO, USA; The one-step PAGE quick preparation kit, SDS-PAGE rapid electrophoresis buffer, rapid transfer buffer, high-efficiency Western blocking buffer, and universal antibody diluent were procured from Shanghai Jifu Life Science Co., Ltd.; PVDF membrane was sourced from Merck, Germany; p-STAT3 antibody, nuclear TET3 antibody, nuclear HOXB2 antibody, c-Myc antibody, NANOG antibody, and NUSAP1 antibody were purchased from Jiangsu Qinke Biological Research Center Co., Ltd.; Goat anti-rabbit IgG (H + L) HRP antibody was obtained from Wuhan Sanying Biotechnology Co., Ltd.; SHP2-OE, TET3-KD, SHP2-KD, and HOXB2-OE lentiviruses were purchased from Shanghai Jiman Biological Company; STAT3 agonist Colivelin Stattic and TET inhibitor TETi76 were purchased from MCE.

Techniques: Over Expression, Protein-Protein interactions, Western Blot, Expressing, Colony Assay, CCK-8 Assay, Standard Deviation

Journal: Scientific Reports

Article Title: SHP2 promotes osteosarcoma via regulating STAT3/TET3/HOXB2 signaling

doi: 10.1038/s41598-026-35493-7

Figure Lengend Snippet: SHP2-OE promotes osteosarcoma progression by upregulating STAT3, TET3, and HOXB2 proteins. ( A ) Experimental results of nude mouse subcutaneous tumor transplantation, including statistical analysis charts for tumor weight and volume. ( B ) Western blot analysis of protein expression bands in tumor tissues, with statistical comparison charts for relative protein expression levels. Mouse groups: NC group, SHP2-OE group, SHP2-OE + TET3-KD group, SHP2-OE + TET3-KD + HOXB2-OE group. ( C ) SHP2 promotes malignant progression of osteosarcoma through the STAT3/TET3/HOXB2 signaling pathway. Data are presented as mean ± SD. N = 6, P < 0.05 indicates statistically significant difference, ns P > 0.05, * P < 0.05, ** P < 0.01.

Article Snippet: Fetal bovine serum (FBS) was purchased from Global Kang Qinhuangdao Biotechnology Co., Ltd.; DMEM basal medium, penicillin-streptomycin solution (double antibiotics), phosphate-buffered saline (PBS), and 0.05% trypsin were obtained from Wuhan Procell Life Science & Technology Co., Ltd.; Matrigel was sourced from Corning, USA; Transwell chambers, dimethyl sulfoxide (DMSO), and SDS-PAGE protein loading buffer were acquired from Shanghai Beyotime Biotechnology Co., Ltd.; High-efficiency RIPA lysis buffer and protease phosphatase inhibitors were purchased from Beijing Solarbio Science & Technology Co., Ltd.; The BCA protein concentration assay kit was obtained from GLPBIO, USA; The one-step PAGE quick preparation kit, SDS-PAGE rapid electrophoresis buffer, rapid transfer buffer, high-efficiency Western blocking buffer, and universal antibody diluent were procured from Shanghai Jifu Life Science Co., Ltd.; PVDF membrane was sourced from Merck, Germany; p-STAT3 antibody, nuclear TET3 antibody, nuclear HOXB2 antibody, c-Myc antibody, NANOG antibody, and NUSAP1 antibody were purchased from Jiangsu Qinke Biological Research Center Co., Ltd.; Goat anti-rabbit IgG (H + L) HRP antibody was obtained from Wuhan Sanying Biotechnology Co., Ltd.; SHP2-OE, TET3-KD, SHP2-KD, and HOXB2-OE lentiviruses were purchased from Shanghai Jiman Biological Company; STAT3 agonist Colivelin Stattic and TET inhibitor TETi76 were purchased from MCE.

Techniques: Transplantation Assay, Western Blot, Expressing, Comparison