Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Design of FR-engineered e23sFv derivatives. (A) Amino acid sequence alignment of the VL and VH domains of mouse anti-HER2 single-chain variable fragment, e23sFv, and their five most homologous counterparts identified in the National Centre for Biotechnology Information protein database. L1-L5 represent VL homologous sequences and H1-H5 represent VH homologous sequences. CDRs and FRs are indicated in columns. The residues that are identical to those of e23sFv are indicated with dashed lines, and missing residues in the CDRs are indicated with asterisks. Non-identical FR residues in e23sFv and all their five homologs in the VL or VH collection are in red. Introduced site-directed mutations are indicated by blue triangles, above which are the corresponding substituted residues. (B) The schematic structure of three e23sFv derivatives. EMEY includes 11 mutated residues in the FRs of e23sFv, as indicated by triangles. EX1 and EX2 represent CDR grafts of e23sFv in the L1-H1 and L2-H2 FR scaffolds, respectively. FR, framework region; VL, light-chain variable region; VH, heavy-chain variable region; CDR, complementarity-determining region.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: In vitro binding of the e23sFv-derived scFvs to recombinant HER2. (A) Affinity measurement by ELISA. HER2-coated microplates were incubated with the e23sFv derivatives at various concentrations, and the bound scFvs were detected using an anti-His antibody. scFv15 served as the negative control. (B) One-shot kinetics of SPR. Five sensorgrams indicated the response of HER2-immobilized sensor chips with five diluted concentrations of the e23sFv derivatives. (C) Comparison of K on , K off and K D of the e23sFv derivatives calculated from SPR sensing. scFv, single-chain variable fragment; SPR, surface plasmon resonance; RU, resonance unit; K on , association rate constant; K off , dissociation rate constant, K D , equilibrium constant; Chi 2 , goodness-of-fit between the binding model and theoretical affinity; OD, optical density.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Vitro, Binding Assay, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, SPR Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Binding of the e23sFv-derived single-chain variable fragments to HER2 on the cell surface. HER2-positive cells (BT-474 and SKOV-3 cells) and HER2-negative cells (MCF-7 cells) were incubated with FITC-labelled e23sFv derivatives and subjected to flow cytometry analysis. (A) Representative dataset of one-parameter histograms. For the isotype control, FITC-labelled scFv15 against HBsAg was used. For the positive control, a commercial FITC-conjugated anti-HER2 antibody was used. (B) Statistical analysis from three independent parallel experiments. * P<0.05; ** P<0.01 and *** P<0.001. ns, non-significant.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Positive Control

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Internalization of the e23sFv-derived single-chain variable fragments by HER2-positive cells. Following incubation with FITC-labelled e23sFv derivatives, BT-474, SKOV-3 and MCF-7 cells were observed under fluorescence microscopy. MCF-7 cells served as the HER2-negative cell controls and scFv15 was the non-specific binding control. Scale bar, 100 µm. The data are representative of at least three independent experiments.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Derivative Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Docking mechanism of the enhanced EX1-HER2 interaction. (A-D) In silico docking of e23sFv, EMEY, EX1 and EX2 and their interactions with the predicted surface models of the HER2 ECD. All the scFv fragments form distinct but overlapping interfaces with domain IV of the HER2 ECD. The 3D structures of (A) e23sFv, (B) EMEY, (C) EX1 and (D) EX2 are presented as coloured ribbons. (E) Binding energy with HER2 and the predicted binding epitopes of all the scFv fragments. ECD, extracellular domain; scFv, single-chain variable fragment.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Silico, Binding Assay

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Brusatol in combination with lapatinib synergistically inhibited the growth of HER2-overexpressed SK-BR-3, SK-OV-3 and AU565 cells (A) SK-BR-3, SK-OV-3 and AU565 cell lines were treated with brusatol, lapatinib or brusatol plus lapatinib in a dose range for 48 h. CCK-8 assays were used to measure the cell viability. Points, mean of 3 independent CCK-8 assays; Bars, SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 (B) The synergistic effect of lapatinib in combination with brusatol was evaluated on the growth of SK-BR-3, SK-OV-3 and AU565 cell line. Combination index (CI) values were calculated using the Chou-Talalay method. Drug synergy, addition, and antagonism are defined by CI values less than 1.0, equal to 1.0, or greater than 1.0, respectively.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: CCK-8 Assay

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Lapatinib plus brusatol abrogate the activation of Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 pathways (A and B) SK-BR-3 and SK-OV-3 cells were treated with lapatinib or brusatol alone, or their combination for 24 h. The changes in Nrf2/HO-1 and EGFR/HER2-AKT/ERK1/2 signaling pathways were monitored by Western Blotting (C and D) Densitometric analysis was performed on the Western Blotting. The levels of Nrf2, HO-1, p-HER2, p-EGFR, p-AKT and p-ERK1/2 were quantified by using the software Image J. The data are expressed as the mean ± SD of three independent experiments. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S4.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Activation Assay, Protein-Protein interactions, Western Blot, Software

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Nrf2 knockdown repressed the activation of HER2 signaling pathway and sensitizes SK-OV-3 cells to lapatinib treatment (A) Effect of Nrf2 knockdown on the expression of HO-1, p-HER2, p-AKT, and p-ERK1/2 were determined after treatment with Nrf2 siRNA or scramble siRNA for 36 h (B) Effect of Nrf2 knockdown on the sensitivity to lapatinib. Cell viability was examined after lapatinib treatment for 36 h in Nrf2 siRNA or scramble siRNA-transfected cells. Cells were transfected with Nrf2 siRNA or scramble siRNA using Lipofectamine 3000 (Invitrogen) according to the supplier's instruction. Data show the mean ± SD (three independent experiments). ∗∗∗ p < 0.001. The original blots were provided in supplementary data as Supplementary Figure S5.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Knockdown, Activation Assay, Expressing, Transfection

Journal: Heliyon

Article Title: The Nrf2 inhibitor brusatol synergistically enhances the cytotoxic effect of lapatinib in HER2-positive cancers

doi: 10.1016/j.heliyon.2022.e10410

Figure Lengend Snippet: Proposed model of the molecular basis of synergistic interaction between lapatinb and brusatol. Brusatol, in combination with lapatinib, may exert synergistic effects in two ways: (a) combination therapy inhibits the phosphorylation of HER receptors including EGFR and HER2, limiting the activation of their downstream pathways including PI3K-AKT signaling and Ras/Raf/MAPK signaling. (b) combination therapy modulates cell redox homeostasis by decreasing Nrf2 level and preventing the accumulation of Nrf2 in the nucleus, interfering its binding to small Maf oncogene family proteins (Mafs) and antioxidant response elements (AREs) complex, causing the inhibition of antioxidant genes such as heme oxygenase 1 (HO-1) and superoxide dismutase (SOD), thereby resulting in ROS accumulation and cell death.

Article Snippet: The antibodies were utilized as following: Nrf2 (1:1000; cat. no. 16396-1-AP; ProteinTech Group, Inc.), HO-1 (1:1000; cat. no. 10701-1-AP; ProteinTech Group, Inc.), HER2 (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-HER2 (Tyr 1221/1222) (1:1,000; cat. no. 2243; Cell Signaling Technology, Inc.), EGFR (1:1,000; cat. no. 54359; Cell Signaling Technology, Inc.), phosphorylated (p)-EGFR (Tyr1068) (1:1,000; cat. no. 2234; Cell Signaling Technology, Inc.), AKT (1:1,000; cat. no. 10176-2-AP; ProteinTech Group, Inc.), p-AKT (Ser 473) (1:2,000; cat. no. 4060; Cell Signaling Technology, Inc.), ERK1/2 (1:2,000; cat. no. 9102; Cell Signaling Technology, Inc.), p-ERK1/2 (Thr202/Tyr204) (1:2,000; cat. no. 8544; Cell Signaling Technology, Inc.), β-actin (1:5,000; cat. no. ab179467; Abcam) and horseradish peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies (1:5,000; cat. no. SA00001-1 or SA00001-2; ProteinTech Group, Inc.).

Techniques: Phospho-proteomics, Activation Assay, Binding Assay, Inhibition

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a , b Stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify novel binding partners. MET wt-tGFP and MET N375S-tGFP cells were labeled with heavy (H) and light (L) amino acids. The cutoff values for SILAC ratios, after normalizing with MET expression, were set at (>2, <0.5) respectively. a Scatter plot of transformed MET wt /MET N375S ratios of membrane-bound proteins. Both axes represent MET wt (H)/MET N375S (L) and MET wt (L)/MET N375S (H) ratios, respectively. b List of various membranous proteins identified in SILAC analysis found to be associated with MET N375S . c Interaction of ectopic MET and endogenous HER2 in H2170 MET wt-tGFP and MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting. Left, input controls. Total MET and HER2 band intensities, normalized to input controls and relative to MET wt , are shown below ( n = 5). d , e Detection of MET/HER2 co-localization (red) in EV, MET wt-tGFP , and MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown ( d ), with the PLA signals quantified ( e ) and expressed as the number of signals/cell ± SD ( n > 3). Scale bar, 20 µm. f Representative confocal microscopy images of MET (Alexa-488; green) and HER2 (Alexa-594; red) in isogenic H2170 clones ( n = 2). DAPI (blue) was used as nuclear counter stain. Co-localized proteins appeared in yellow. The smaller panels are detailed views of the outlined (white) squares in the respective images. Scale bar, 20 µm. The total MET fluorescence that co-localized with HER2 signal in each variant was tabulated on the right, data presented as mean ± SD (five fields in one representative experiment). g MET/HER2 interaction in H2170 MET wt-tGFP and MET N375S-tGFP tumors shown in Fig. was detected with immunoprecipitation and immunoblotting. IgG was used as a loading control. h – k The role of Sema domain in MET N375S-tGFP cells was examined with recombinant Sema proteins, wild-type, rSema wt ; N375S mutant, rSema N375S . Cell viability of MET wt-tGFP ( h ) and MET N375S-tGFP ( i ) cells after treatment with 1, 5, 10 µg/ml of rSema wt or rSema N375S for 72 h, presented as mean ± SD ( n = 3). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. j Immunoblots showing the total and phosphorylated expressions of MET, HER2, Src, Akt, and ERK1/2 in lysates of the indicated cell lines after treatment with 10 µg/ml rSema. β-Actin was used as a loading control. k MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected with immunoprecipitation and immunoblotting after treatment with 10 µg/ml rSema. Left, input controls. l MET/HER2 interaction in H2170 MET N375S-tGFP cells was detected after serum starvation (0.1% FBS), or co-incubated with HGF (0.1% FBS + 10 ng/ml HGF). Left, input controls. m HEK293 cells were transfected with 1 µg of either pCMV6-EV-tGFP vector, MET-wt, N375S, ∆Sema, N375Q, M1268T, or Y1248H plasmid, together with pCMV6-ERBB2-DDK plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to MET wt , are shown below ( n = 3). Left, input controls and phosphorylated proteins. β-Actin was used as a loading control.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Quantitative Proteomics, Cell Culture, Multiplex sample analysis, Binding Assay, Labeling, Expressing, Transformation Assay, Membrane, Immunoprecipitation, Western Blot, Proximity Ligation Assay, Confocal Microscopy, Clone Assay, Staining, Fluorescence, Variant Assay, Control, Recombinant, Mutagenesis, Two Tailed Test, Incubation, Transfection, Plasmid Preparation

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Isogenic H2170 MET N375S-tGFP cells were treated with vehicle (0.1% DMSO), MET inhibitor crizotinib (1 µM), HER2 inhibitor lapatinib (0.3 µM), or crizotinib/lapatinib combination. a Interaction of ectopic MET and endogenous HER2 was detected with immunoprecipitation and immunoblotting. Total MET and HER2 band intensities, relative to vehicle treated group, are shown below. Values represent average of three independent experiments. Left, input controls. b Detection of MET/HER2 co-localization (red) in MET N375S-tGFP cells with proximity ligation assay (PLA). Representative images are shown. Scale bar, 20 µm. c The PLA signals were quantified and expressed as number of signals/cell ± SD ( n > 3). Crizo, crizotinib; Lapa, lapatinib. d MET/HER2 interaction in H2170 MET N375S-tGFP tumors from Supplementary Fig. was detected with immunoprecipitation and immunoblotting after crizotinib treatment. Left, input controls. Cells were harvested 48 h after treatment, β-Actin was used as a loading control. e – h Immunohistochemistry staining of H2170 MET wt-tGFP and MET N375S-tGFP tumors from Supplementary Fig. . Representative images for p-MET ( e ) and p-HER2 ( g ) staining are shown. Expression of p-MET ( f ) and p-HER2 ( h ) were quantified and expressed as mean of positive-staining/100 cells ± SD ( n = 5). Scale bar, 50 µm. Two-tailed Student’s t test; * P < 0.05.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Immunoprecipitation, Western Blot, Proximity Ligation Assay, Control, Immunohistochemistry, Staining, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: A common MET polymorphism harnesses HER2 signaling to drive aggressive squamous cell carcinoma

doi: 10.1038/s41467-020-15318-5

Figure Lengend Snippet: a – c Cellular invasion on isogenic wild-type (MET wt-tGFP ) and N375S mutant (MET N375S-tGFP ) clones, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. a H2170 MET N375S-tGFP cells were treated with crizotinib (10 µM), lapatinib (0.3 µM) or a crizotinib/lapatinib combination for 36 h. Representative images of cell invasion are shown. b H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 36 h. Representative images are shown. Scale bar: 200 μm. c Percentage of invaded cells relative to vehicle control (0.1% DMSO) or Scr siRNA control were expressed as mean ± SD ( n = 3). One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. Left, immunoblots of siRNA-treated H2170 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. d – f Calu-1 MET wt-tGFP and MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in Matrigel invasion chambers for 24 h. Representative images of cell invasion for MET wt-tGFP ( d ) and MET N375S-tGFP cells ( e ) are shown. Scale bar: 200 μm. f Percentage of invaded cells relative to Scr siRNA control in Calu-1 MET wt-tGFP and MET N375S-tGFP cells were expressed as mean ± SD ( n = 3). Left, immunoblots of siRNA-treated Calu-1 MET N375S-tGFP cells. For immunoblots, cells were harvested 48 h after siRNA treatment, and β-actin was used as a loading control. One-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. g , h Anchorage-independent colony formation on isogenic H2170 MET N375S-tGFP cells, treated with kinase inhibitors or siRNA silencing of MET or HER2, were being evaluated. g H2170 MET N375S-tGFP cells were co-incubated with 10 nM of the indicated siRNA overnight, and seeded in soft agar for 4 weeks. Representative images are shown. h The number of colonies were quantified after treatment with crizotinib, lapatinib, trastuzumab, or in combination. Data were presented as percentage of colonies relative to vehicle control (0.1% DMSO) ± SD ( n = 3). i – l Efficacies of HER2 inhibitors were evaluated in xenograft models. Tumor growth of MET wt-tGFP ( i ) and MET N375S-tGFP ( j ) xenografts after treatment with trastuzumab, pertuzumab, lapatinib, and ASLAN001 were expressed at mean ± SEM ( n = 5). Two-way ANOVA; * P < 0.05, ** P < 0.01, *** P < 0.001. k Immunohistochemistry staining showing the changes in p-MET after treatment with trastuzumab in MET wt-tGFP and MET N375S-tGFP tumors. Representative images are shown. Scale bar, 50 µm. l Expression of p-MET was quantified and expressed at mean of positive-staining/100 cells ± SD ( n = 5). Two-tailed Student’s t test; * P < 0.05, ** P < 0.01, *** P < 0.001. m HEK293 cells were transfected with 1 µg of either pCMV6-ERBB2-wt, ∆D1, ∆D2, ∆D3, ∆D4, or ∆TK plasmid, together with pCMV6-MET-N375S plasmid, for 24 h. MET/HER2 interaction in HEK293 cells was detected with immunoprecipitation and immunoblotting. Left, input controls and phosphorylated proteins. β-Actin was used as a loading control. n , o Isogenic Calu-1 MET wt-tGFP ( n ) and MET N375S-tGFP ( o ) cells were engrafted into SCID mice, and treated daily with vehicle ( n = 6) or 15 mg/kg afatinib ( n = 5) starting 14 days after inoculation. Arrows indicate treatment start date. Kaplan–Meier analyses of the mice are shown.

Article Snippet: For immunoblotting, p-MET (Tyr1234/1235, #3077), total MET (#8198), p-HER2 (Tyr1221/1222, #2243), total HER2 (#2165), p-p38 (Thr180/Tyr182, #4511), total p38 (#8690), p-Src (Tyr416, #2101), total Src (#2108), p-ERK1/2 (Thr202/Tyr204, #4377), total ERK1/2 (#4695), p-mTOR (Ser2448, #2971), total mTOR (#2172), p-Stat3 (Tyr705, #9131), total Stat3 (#4904), p-EGFR (Tyr1068, #2234), total EGFR (#2646), p-Akt (Ser473, #4060), total Akt (#9272), α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated MET (#24294), HER2 (#60388), HSP90 (#4874), and β-actin (#5125) antibodies were purchased from Cell Signaling Technology; turboGFP antibody was from Origene (#TA150041); pan-Cadherin antibody (#ab22744) from Abcam.

Techniques: Mutagenesis, Clone Assay, Incubation, Control, Western Blot, Immunohistochemistry, Staining, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Immunoprecipitation

Journal: Nature Communications

Article Title: Mismatch repair deficiency predicts response to HER2 blockade in HER2-negative breast cancer

doi: 10.1038/s41467-021-23271-0

Figure Lengend Snippet: A Western blots demonstrating increase in pHER2 and downstream signaling in sh MLH1 MCF7 cells treated with fulvestrant relative to sh Luc cells. Quantification of four independent replicates conducted through ImageJ in accompanying dot plots. Validation in T47D cells in Fig. . Immunofluorescent staining for HER2 in MCF7 sh Luc and sh MLH1 cells in vitro ( B ), in MCF7 sh Luc and sh MLH1 xenograft tumors ( C ), and in WHIM20, PMS2 mutant, ER + /HER2 − PDX tumors ( D ), grown with or without fulvestrant. Accompanying quantification presented as strip charts. Three independent experiments or tumors from each group were quantified. Two-sided Student’s t test determined p values. Supporting data from FACS analysis are presented in Fig. . Scale bars represent 50 µ. Source data for all figures available with paper.

Article Snippet: Antibodies used were pHER2 Y1196 (D66B7) (Cell Signaling; cat# 6942S), total HER2 (Thermo Scientific; NeoMarkers; cat# MS-730-P1ABX), pAkt S473 (D9E) XP (Cell Signaling; cat#4060S), total Akt (Cell Signaling; cat#9272S), pS6 (S235/236) (Cell Signaling; cat# 2211S), total S6 (5G10) (Cell Signaling; cat# 2217S), MLH1 (1:2,000, Sigma-Aldrich; cat# WH0004292M2), ER clone 60C (EMD Millipore; cat# 04-820), and GAPDH (0411) (Santa Cruz; cat# sc-47724).

Techniques: Western Blot, Biomarker Discovery, Staining, In Vitro, Mutagenesis, Stripping Membranes

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Rapid internalization of anti-HER2-Bs compared with trastuzumab or 39S in BT-474 cells. Internalization time courses of trastuzumab-AF647 ( A – C ), 39S-AF647 ( D – F ), and anti-HER2-Bs-AF647 ( G – I ) in cells stained with cytoplasmic dye, CFSE) (green), are shown. Before the initiation of internalization, fluorescence antibodies are localized on the cell surface ( A , D , E ). At 30 min, both trastuzumab-AF647 (red) and 39S-AF647 (red) are slowly internalized into the cells ( B , E ), while a majority of anti-HER2-Bs-AF647 (red) are already internalized ( H ). At 2 h, the surface-bound anti-HER2-Bs-AF647 is completely internalized ( I ), while both trastuzumab-AF647 and 39S-AF647 remain on the cell surface ( C , F ). ( J ) Antibody internalization time course was quantified by an image analysis algorithm. Data are plotted as mean ± standard deviation from 6 wells. Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Staining, Fluorescence, Standard Deviation

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Effect of dynamin and clathrin inhibitors on anti-HER2-Bs and trastuzumab internalization. Overlays of anti-HER2-Bs-AF647 ( A – F ) and trastuzumab-AF647 ( G – L ) with the cytoplasm dye (green) in BT-474 cells treated with either DMSO ( A , D , G , J ), dynamin inhibitor Dyngo 4a ( B , E , H , K ), or clathrin light chain inhibitor Pitstop 2 ( C , F , I , L ) are shown. At T = 0, anti-HER2-Bs-AF647 ( A , B , C ) or trastuzumab-AF647 ( G , H , I ) was bound on the surface of the treated cells. At 2 h, the internalization of both anti-HER2-Bs-AF647 (red) and trastuzumab-AF647 (red) was inhibited in the presence of Dyngo 4a (compare B , E to A , D for anti-HER2-Bs; compare H , K to G , J for trastuzumab) or Pitstop 2 (compare C , F to A , D for anti-HER2-Bs; compare I , L to G , J for trastuzumab). Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: The combination of antibodies did not accelerate internalization. However, the addition of a secondary antibody accelerated trastuzumab internalization. Combined treatment with trastuzumab-AF647 (red) and unlabeled 39S ( C ) did not accelerate internalization of trastuzumab compared to trastuzumab-AF647 alone at 1 h ( A ). The internalization rate of trastuzumab-AF647 in the presence of 39S ( C ) is similar to the single antibody treatment with trastuzumab-AF647 ( A ) or 39S-AF647 (red) ( B ). By contrast, and similar to the previous figures, anti-HER2-Bs-AF647 (red) internalized rapidly into the cells ( E ). ( D ) Addition of anti-human IgG accelerated the internalization of trastuzumab compared to the untreated cells (compare D to A ). However, the internalization rate of anti-HER2-Bs is still much more pronounced compared to the trastuzumab-AF647/anti-human IgG-treated cells (compare E to D ). Cytoplasm dye, CFSE, is shown in green. Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Blocking one epitope on HER2 receptors slowed down the internalization of anti-HER2-Bs-AF647 significantly. Cytoplasm dye, CFSE, is shown in green. Similar to the previous figures shown, anti-HER2-Bs-AF647 (red) internalized rapidly ( C ) compared to either trastuzumab-AF647 (red) ( A ) or 39S-AF647 (red) ( B ) at 2 h. ( D ) By contrast, blocking of trastuzumab-binding epitopes significantly slowed down the internalization of anti-HER2-Bs-AF647 at 2 h (compare D to C ) and resembled the internalization of 39S alone (compare D to B ). ( E ) Similarly, blocking of the other epitopes (39S-binding epitopes) also slowed down the anti-HER2-Bs-AF647 internalization (compare E to C ). Note that at the start of internalization (T = 0), similar fluorescent signals (AF647) were observed for all tested conditions ( A – E ) (see ). Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Blocking Assay, Binding Assay

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs, but not trastuzumab, 39S, or both in combination, induced efficient HER2 degradation. ( A ) The level of total HER2 was not changed in the cells treated with trastuzumab, 39S, or both in combination. However, treatment with anti-HER2-Bs resulted in a decrease in total HER2 expression by 6 h. ( B ) Anti-HER2-Bs induces HER2 receptor ubiquitination, and this coincides with HER2 degradation. Treatment with anti-HER2-Bs resulted in an increase in total HER2 ubiquitination at 1–2 h and K63-specific ubiquitination at 1 h. ( C ) Quantification of HER2 total ubiquitination level normalized to total HER2 receptor level. The normalized level of HER2 total ubiquitination increased from 30 min post treatment and was sustained during 2–6 h post treatment.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Expressing

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs and trastuzumab mediated distinct intracellular trafficking of internalized HER2 receptors. Internalization time courses of anti-HER2-Bs-AF647 ( A – C ) and trastuzumab-AF647 ( D – F ) are shown. ( A , D ) Before the initiation of internalization, both anti-HER2-Bs-AF647 and trastuzumab-AF647 (red) were colocalized with HER2 receptors on the cell surface (green) and appeared yellow. ( B , E ) At 30 min, the antibody-HER2 complexes (appearing as yellow puncta) were internalized into the cells. Consistent with the previous figures, the internalization of anti-HER2-Bs-AF647 is more rapid compared to trastuzumab-AF647 (compare D to G ). ( C , F ) Anti-HER2-Bs-AF647 and trastuzumab-AF647 mediated strikingly different trafficking of HER2 receptors at 2 h. ( C ) Internalized anti-HER2-Bs-AF647 (red) remained colocalized with HER2 receptors (green) and appeared as yellow puncta. However, trastuzumab-AF647 was dissociated from HER2 receptors and appeared as red puncta in the cells ( F ). ( G , H ) Treatment with anti-HER2-Bs, but not trastuzumab, resulted in complete surface clearance of HER2 receptors at 8 h. ( I , J ) Internalized anti-HER2-Bs-AF647 and trastuzumab-AF647 are colocalized with a lysosomal marker, LAMP1-AF488, at 8 h. The puncta of internalized anti-HER2-Bs-AF647 ( I ) or trastuzumab-AF647 ( J ) (red), colocalized with the LAMP1-AF488-positive compartments (green) merging in yellow. Treatment with anti-HER2-Bs resulted in the complete clearance of the antibody from the cell surface by 8 h ( I ). By contrast, there was a significant amount of trastuzumab remaining on the cell surface ( J ). Scale bars are 10 μm. ( K ) Treatment with anti-HER2-Bs resulted in a ~93% reduction of HER2 ectodomain (ECD) shedding in the conditioned culture media compared to the untreated cells. Treatment with trastuzumab also reduced HER2 ECD shedding by ~76%. ( L , M ) In addition, treatment with anti-HER2-Bs also reduced the number of exosome particles and the level of HER2 expression in the normalized exosomal fraction. CD63 is the protein marker for exosomes purified from conditioned culture media.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Marker, Expressing, Purification

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: A proposed model of anti-HER2-Bs- and trastuzumab-mediated internalization, trafficking, and the fate of HER2 receptors. Please note that the receptors are drawn unproportionally to highlight the crosslinking on the cell surface and the intracellular trafficking. ( A ) In the untreated cells, the HER2 receptors are predominately localized on the cell surface. As part of the normal turnover process, a basal level of HER2 continuously undergoes internalization and catabolism. The pool of cell surface HER2 is replenished by the de novo synthesis of HER2 receptors. The internalized HER2 is then trafficked to multivesicular bodies (MVBs) for subsequent sorting. The HER2-loaded MVBs can fuse either with lysosomes for degradation, or with plasma membrane to release intra-luminal vesicles (ILVs) as exosomes. In addition, HER2-ECDs are constantly shedding from the receptors on the cell surface. ( B ) Treatment with trastuzumab resulted in a slow internalization of HER2 receptors via a clathrin-mediated pathway. The internalized trastuzumab-HER2 complexes are dissociated in the endosomes. The dissociated HER2 receptors are then recycled back to the cell surface. Similar to the untreated cells, a basal level of HER2 is internalized and trafficked to MVBs as a process of normal turnover, leading to either degradation of HER2 or release of HER2-loaded exosomes. On the other hand, the dissociated trastuzumab is trafficked to MVBs and lysosomes for degradation. Treatment with trastuzumab did not affect the level of exosome formation. However, binding of trastuzumab to the domain IV of the HER2 receptors on the cell surface inhibits the cleavage of HER2-ECD. ( C ) Concurrent engagement of HER2 epitopes by the bivalent bispecific anti-HER2-Bs antibody may induce crosslinking of HER2 receptors to form high molecular weight complexes on the cell surface. The anti-HER2-Bs-HER2 complexes are internalized rapidly via a clathrin-mediated pathway. The anti-HER2-Bs-HER2 complexes stay associated and traffic through the endocytic pathway. Instead of being recycled back to the cell surface, anti-HER2-Bs induces redirected trafficking of HER2 receptors to lysosomes for degradation. The degradation of HER2 receptors coincides with an increase in HER2 ubiquitination (total and K63). As a result of the lysosomal targeting, the anti-HER2-Bs-HER2-loaded multivesicular bodies are not able to fuse with the cell membrane, and thus this results in reduced exosomal export. In addition, rapid internalization of surface anti-HER2-Bs-HER2 complexes results in a fast clearance of surface HER2 receptors, thus leading to a reduction of HER2 ECD shedding on the cell surface.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Binding Assay, Molecular Weight