Journal: Frontiers in Oncology

Article Title: A trispecific antibody targeting EGFR/cMET/VEGF-A demonstrates multiple mechanisms of action to inhibit wild-type and mutant NSCLC animal models

doi: 10.3389/fonc.2025.1533059

Figure Lengend Snippet: TAVO412 bound to NSCLC cell lines and blocked binding of EGF and HGF. The binding of TAVO412 (red open circle), Amivantamab analogue (blue open circle) and null mAb (black open circle) to (A) NCI-H292; (B) HCC827; and (C) NCI-H1975 NSCLC cell lines as analyzed by flow cytometry. See Supplementary Table S2 for corresponding EC 50 , 95% CI for EC 50 , and efficacy (span in y axis). Blocking of (D) EGF and (E) HGF from binding to HCC827 cells was assessed by flow cytometry with TAVO412 (red open circle), Amivantamab analogue (blue open circle) and null mAb (black open circle). See Supplementary Table S3 for corresponding IC 50 , 95% CI for IC 50 , and efficacy (span in Y-axis). The data from three independent experiments were expressed as the mean ± SEM of duplicate treatments. The amivantamab analogue served as a positive control molecule while the null mAb served as a negative control. The abbreviations were: gMFI, geometric mean fluorescent intensity; AF647, Alexa Fluor 647 dye; AF488, Alexa Fluor 488 dye; Ab, antibody; nM, nanomolar; EGF, epidermal growth factor; HGF, hepatocyte growth factor; SEM, standard error of the mean.

Article Snippet: Rabbit anti-human EGF antibody (Sino Biological, #10605-T16) and Alexa Fluor 488 (AF488)-labeled anti-rabbit IgG1 (Jackson ImmunoResearch, #111-545-144) were added to test for EGF binding, while AF488 streptavidin (Invitrogen, # S11223 ) was added to test HGF binding.

Techniques: Binding Assay, Flow Cytometry, Blocking Assay, Positive Control, Negative Control

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Human normal ovary, ovarian tumor, and healing wound tissues were dissociated, and isolated endothelial cells and samples were processed for microarray. b, Gene microarray of endothelial cells from normal ovary, healing-wound tissue, and ovarian tumor–associated endothelial cells. c, Expression of EGFL6 in human normal ovary, wound, and ovarian tumor samples. Representative images were taken from different samples. Scale bar =100 µm d, Validation of gene microarray data using q-PCR.(Normal ovary; n = 5, Ovarian tumor; n = 10, Wound; n = 7). Validation Error bars indicates SEM. *p<0.05 vs. Normal. See also Figure S1.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Isolation, Microarray, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Graph of wound area on mice treated with either control IgG antibody or DC101 (anti-VEGFR2) quantified on days 0 through 15 after a skin excision wound. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control IgG. b, One day after SKOV3ip1 tumor cell injection, a wound was created on the dorsal side of the mice. Animals were treated with either Control siRNA-CH or mEGFL6 siRNA-CH. Graphical depiction of wound areas quantified on days 0 through 15 after skin excision wound. c, Effect of mEGFL6 silencing on tumor growth; representative images of tumor burden. Tumor weights are shown in d and numbers of tumor nodules in e. (n=10 mice per group); error bars indicate SEM. *p<0.05 vs. Control siRNA. f, Hind limb ischemia. After arterial ligation, mice were separated into 3 groups (n = 5 mice per group): normal, ischemia-24 h, and 96 h. Blood flow was monitored before and after femoral artery ligation with use of serial color doppler. At each time point, tissue was harvested and frozen so that immunofluorescence staining could be performed. g, EGFL6 expression was increased in endothelial cells in the ischemic (hypoxic) condition compared with the normal conditions. Error bars indicate SEM. *p<0.05 vs. Normal. See also Figure S2.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Injection, Ligation, Immunofluorescence, Staining, Expressing

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, EGFL6 promoter reporter analysis under normoxia and hypoxic conditions. b, TWIST1 ectopic expression increases EGFL6 transcription activity. c, Increase in TWIST1 and EGFL6 expression in hypoxia and CoCl2 treatment. d, Ectopic expression of TWIST1 increases EGFL6 expression in RF24 cells. e, ChIP analysis of TWIST1 binding to EGFL6 promoter region in hypoxia compared with normoxia. Cross-linked chromatin from RF24 cells incubated in hypoxia chamber for 48 h and immunoprecipitated with TWIST1 or IgG control antibodies. The input and immunoprecipitated DNA was subjected to PCR using primers corresponding to the base pairs upstream of EGFL6 transcription start site. f, EGFL6 gene silencing using siRNA leads to increased cell death in hypoxia condition. (n = 3) **p<0.005, *p<0.05 See also Figure S3.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Expressing, Activity Assay, Binding Assay, Incubation, Immunoprecipitation

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Expression level change in selected proteins after normalization by RPPA analysis. b and c, Western blotting of EGFL6-mediated activation of PI3K/AKT signaling, Tie2 and EGFR signaling. d, RF24 cells treated with Control (PBS) or EGFL6. Extracts were subjected to anti-Tie2 immunoprecipitation (IP) and integrin α5, αV, β1, and β3 detected by immunoblotting. e, Expression level of pTie2 and pAKT in cytosol and membrane fractions with Control (PBS) and EGFL6 treatment. αβ-tubulin was used as internal control of cytosolic fraction; NaK ATPase was used as membrane marker. f, Silencing of Integrin β1 (ITGB1) and Tie2 using specific siRNAs decreases Tie2 and AKT signaling. g–h, Silencing of Integrin β1 (ITGB1) and Tie2 decreases EGFL6-mediated tube formation (g) and migration (h) in endothelial cells. *p<0.05 vs. Control siRNA. In i–k, RGD blocking peptide decreases Tie2/AKT activation (i), tube formation (j) and migration (k). (n=3) *p<0.05 vs. Control. See also Figure S4.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Expressing, Western Blot, Activation Assay, Immunoprecipitation, Marker, Migration, Blocking Assay

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, The binding affinity of recombinant EGFL6 to monoclonal antibody #93 and #135 was measured by Biacore. The dissociation constant (Kd) value of the monoclonal antibodies were calculated to be 1.89 nM (#93) and 2.19 nM (#135). b, Effect of EGFL6 blocking antibodies on Tie2/AKT activation in RF24 cells (n=3). c, Effect of EGFL6 blocking antibodies on wound healing in vivo (n=5 mice/group, #135; 5 mg/kg). d and e, EGFL6 antibodies decreased tube formation and migration of RF24 cells. f, Effect of EGFL6 blocking antibodies on tumor weight and tumor nodules in SKOV3ip1 tumor-bearing mice. Seven days after tumor cell injection, mice were randomly divided into three groups (10 mice/group) to receive therapy: (1) Control IgG Ab, (2) EGFL6 #93, and (3) EGFL6 Ab #135 (5 mg/kg). Antibody treatment was given once a week. g, Effect of targeted EGFL6 on proliferation (Ki67) and microvessel density (CD31). Scale bar = 100µm. The bars in the graphs correspond sequentially to the labeled columns of images on the left. Error bars indicates SEM. *p<0.05 vs. Control IgG. See also Figure S5.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Binding Assay, Recombinant, Blocking Assay, Activation Assay, In Vivo, Migration, Injection, Labeling

Journal: Cell reports

Article Title: Differential effects of EGFL6 on tumor versus wound angiogenesis

doi: 10.1016/j.celrep.2017.11.020

Figure Lengend Snippet: a, Tumor growth in Tie2;EGFL6 KO mice (KO) and WT mice. ID8 murine ovarian cancer cells were injected into KO and WT mice (n=10 mice per group). b, Double immunofluorescence staining of CD31 and EGFL6 in ID8 tumor from WT and KO mice. Scale bar = 100 µm. c, Bar graph represents tumor weight. d and e, Proliferation (Ki67) and microvessel density (CD31) counted in WT and KO mice tumor sections. Error bars indicate SEM. Scale bar = 100 µm. *p<0.05 vs. WT mice. f, Representative images of human ovarian cancer vasculature with low or high immunohistochemical staining for EGFL6. Scale bar =200 µm. Representative images were taken from different samples. g, Kaplan-Meier curves of disease-specific mortality of patients whose ovarian vasculature expressed low versus high EGFL6. See also Figure S6 and Table S1.

Article Snippet: Generation of EGFL6 monoclonal antibody Anti-EGFL6 monoclonal antibodies were identified by screening a large panel of single memory B cells collected from rabbits after immunization with recombinantly expressed human EGFL6 protein (SinoBiological, Beijing, China).

Techniques: Injection, Double Immunofluorescence Staining, Immunohistochemical staining, Staining

Journal: Oncotarget

Article Title: The HGF inhibitory peptide HGP-1 displays promising in vitro and in vivo efficacy for targeted cancer therapy

doi:

Figure Lengend Snippet: a. K D of HGP-1 binding to HGF was determined by SPR technique. b. The assessment of binding competition between various proteins and HGF by fluorescence-based ELISA assay post 1.5-hour incubation. Proteins at the concentrations of 0.05 nM, 0.5 nM, 5 nM and 50 nM mixed with 10 μM FITC-labeled HGP-1 were the liquid phase ( n = 5). c. The binding activity between HGP-1 to HGF and EGF were measured by fluorescence-based direct ELISA assay post 1.5-hour incubation. HGP-1 at the concentrations of 0.1 μM, 1 μM, 10 μM, 100 μM were used ( n = 3). Values were mean ± SEM.

Article Snippet: Recombinant human full length HGF (10463- NHAC-A), VEGF-165 (11066-HNAB), Protein G (13103-PNAE), Biotinylated recombinant human full length HGF (10463-HNAC-B), HGF antibody (10463-RP01-B), MET antibody (10692-MM02), EGF antibody (0605-R008) were purchased from Sino Biological Inc. Recombinant human EGF (H6000–10), bFGF (H3000–10) were obtained from Beijing Wishbiotechnology Co., Ltd. Dynabeads MyOne streptavidin C1 magnetic beads (#65002) and Streptavidin Phycoerythrin Conjugates (S-866) were obtained from Invitrogen.

Techniques: Binding Assay, Fluorescence, Enzyme-linked Immunosorbent Assay, Incubation, Labeling, Activity Assay, Direct ELISA

Journal: Oncology Reports

Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

doi: 10.3892/or.2017.6025

Figure Lengend Snippet: EGFR expression in seventeen cell lines measured by western blotting. The cell lines were 7402, 7703, 7721, Hep3B, HepG2, H460, SPCA1, SKOV-3, HeLa, MCF-7, glioma cells U87, U251, C33a, PC-3, NCI-H1299, NCI-H446, and K562 cells. Hep3B, HeLa, PC-3, NCI-H1299, and NCI-H466 cells exhibited higher expression levels of EGFR than those of other cell lines. Combined with the results presented in , HeLa cells had high expression of both HIP1 and EGFR.

Article Snippet: After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times.

Techniques: Expressing, Western Blot

Journal: Oncology Reports

Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

doi: 10.3892/or.2017.6025

Figure Lengend Snippet: Expression levels of EGFR in HeLa cells with HIP1 Stealth RNAi. Neither siRNA1 nor siRNA2 had an effect on the expression levels of EGFR and GAPDH in HeLa cells.

Article Snippet: After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

doi: 10.3892/or.2017.6025

Figure Lengend Snippet: Expression levels of EGFR in NCI-H1299 cells after HIP1 Stealth RNAi. siRNAs had no effect on the expression of EGFR or GAPDH in NCI-H1299 cells.

Article Snippet: After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

doi: 10.3892/or.2017.6025

Figure Lengend Snippet: Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (1.5 ng/ml EGF stimulation). After 1.5 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.

Article Snippet: After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times.

Techniques: Expressing

Journal: Oncology Reports

Article Title: Knockdown of HIP1 expression promotes ligand-induced endocytosis of EGFR in HeLa cells

doi: 10.3892/or.2017.6025

Figure Lengend Snippet: Endocytosis of EGFR and localization of clathrin in HeLa and HeLa HIP1 RNAi2 cells (100 ng/ml EGF stimulation). After 100 ng/ml EGF stimulation, endocytosis of EGF-bound EGFR and clathrin were accelerated significantly after the expression of HIP1 was blocked. EGFR and clathrin were colocalized in the cytoplasm.

Article Snippet: After SDS-PAGE, the protein was transferred to a nitrocellulose membrane using a semidry transfer apparatus (Bio-Rad, Hercules, CA, USA), blocked with 3% bovine serum albumin in TBST [10 mmol/l Tris (pH 8.0), 150 mmol/l NaCl, 0.1% Tween-20], incubated at room temperature for 1 h with rabbit anti-human HIP1, EGFR, and GAPDH antibodies, rotated for 1 h at room temperature of 25°C, and washed with TBST three times.

Techniques: Expressing