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rabbit anti human erbb2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti human erbb2
    LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent <t>ERBB2</t> (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.
    Rabbit Anti Human Erbb2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 250 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rabbit+anti+human+her2+mab/HER2%2FErbB2+XP+Rabbit+mAb/pmc12576823-241-33-36
    Average 95 stars, based on 250 article reviews
    rabbit anti human erbb2 - by Bioz Stars, 2026-09
    95/100 stars

    Images

    1) Product Images from "Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery"

    Article Title: Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.5c15047

    LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent ERBB2 (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.
    Figure Legend Snippet: LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent ERBB2 (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.

    Techniques Used: Western Blot, Control, Two Tailed Test

    Related Articles

    Staining:

    Article Title: Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance
    Article Snippet: .. The tumor sections were stained using rabbit anti-human HER2 mAb (Cell Signaling) and an immunohistochemistry application solution kit (Cell signaling, catalog #: 13079) according to the manufacturer’s manual. .. Finally, bright field images (20X and 40X) were taken using an Invitrogen EVOS-FL Auto 2 microscope.

    Article Title: Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance
    Article Snippet: .. The tumor sections were stained using rabbit anti-human HER2 mAb (Cell Signaling, catalog number: 2165S, diluted 1 : 200) and an IHC application solution kit (Cell Signaling, catalog number: 13079) according to the manufacturer’s manual. .. Finally, bright-field images (×20 and ×40) were taken using an Invitrogen EVOS-FL Auto 2 microscope.

    Immunohistochemistry:

    Article Title: Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance
    Article Snippet: .. The tumor sections were stained using rabbit anti-human HER2 mAb (Cell Signaling) and an immunohistochemistry application solution kit (Cell signaling, catalog #: 13079) according to the manufacturer’s manual. .. Finally, bright field images (20X and 40X) were taken using an Invitrogen EVOS-FL Auto 2 microscope.

    Article Title: Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance
    Article Snippet: .. The tumor sections were stained using rabbit anti-human HER2 mAb (Cell Signaling, catalog number: 2165S, diluted 1 : 200) and an IHC application solution kit (Cell Signaling, catalog number: 13079) according to the manufacturer’s manual. .. Finally, bright-field images (×20 and ×40) were taken using an Invitrogen EVOS-FL Auto 2 microscope.

    Western Blot:

    Article Title: Cellular Receptor Tyrosine Kinase Signaling Plays Important Roles in SARS-CoV-2 Infection
    Article Snippet: Total protein was subjected to 8% SDS-PAGE gel, blocked with 5% BSA, and then blotted onto a nitrocellulose membrane (Thermo Scientific, #88018, Waltham, MA, USA) using the transturbo semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). .. The following antibodies were used for immunoblotting: rabbit anti-human TrkA polyclonal antibody (Cell Signaling Technology, 2505S, Danvers, MA, USA), rabbit anti-human pTrkA monoclonal antibody (mAb) (Cell Signaling Tech, 4168S), rabbit anti-human HER2 mAb (Cell Signaling Tech, 2165S), rabbit anti-human pHER2 mAb (Cell Signaling Tech, 2243L), mouse anti-human EGFR mAb (Cell Signaling Tech, 2239S), rabbit anti-human pEGFR mAb (Cell Signaling Tech, 3777S), rabbit anti-human GAPDH mAb (Cell Signaling Tech, 2118S), anti-rabbit HRP conjugated (Bio Rad, 1706515, Hercules, CA, USA), and anti-mouse IgG HRP conjugated (R&D, HAF007). .. After immunoblotting, the membrane was applied with Immobilon Western Chemiluminescent HRP Substrate (MilliporeSigma, WBULP, Burlington, MA, USA) for imaging on an iBright FL1500 system (Thermo Fisher Scientific).

    Article Title: Cellular Receptor Tyrosine Kinase Signaling Plays Important Roles in SARS-CoV-2 Infection
    Article Snippet: Total protein was subjected to 8% SDS-PAGE gel, blocked with 5% BSA, and then blotted onto a nitrocellulose membrane (Thermo Scientific, #88018, Waltham, MA, USA) using the trans-turbo semi-dry transfer apparatus (Bio-Rad, Hercules, CA, USA). .. The following antibodies were used for immunoblotting: rabbit anti-human TrkA polyclonal antibody (Cell Signaling Technology, 2505S, Danvers, MA, USA), rabbit anti-human pTrkA monoclonal antibody (mAb) (Cell Signaling Tech, 4168S), rabbit anti-human HER2 mAb (Cell Signaling Tech, 2165S), rabbit anti-human pHER2 mAb (Cell Signaling Tech, 2243L), mouse anti-human EGFR mAb (Cell Signaling Tech, 2239S), rabbit anti-human pEGFR mAb (Cell Signaling Tech, 3777S), rabbit anti-human GAPDH mAb (Cell Signaling Tech, 2118S), anti-rabbit HRP conjugated (Bio Rad, 1706515, Hercules, CA, USA), and anti-mouse IgG HRP conjugated (R&D, HAF007). .. After immunoblotting, the membrane was applied with Immobilon Western Chemiluminescent HRP Substrate (MilliporeSigma, WBULP, Burlington, MA, USA) for imaging on an iBright FL1500 system (Thermo Fisher Scientific).

    other:

    Article Title: Antibody-drug conjugates with dual payloads for combating breast tumor heterogeneity and drug resistance
    Article Snippet: The other antibodies used in this study were purchased from commercial vendors as follows: Mouse anti-MMAE/F mAb (LEV-MAF3) from Levena Biopharma; goat anti-human IgG Fab-horseradish peroxidase (HRP) conjugate (109-035-097), goat anti-human IgG Fc antibody (109-005-098), donkey anti-human IgG-HRP conjugate (709-035-149), and goat anti-mouse IgG-HRP conjugate (115-035-071) from Jackson ImmunoResearch; mouse anti-human ERBB2 (CD340, HER2) Vio® Bright FITC (130-121-436) from Miltenyi Biotec; and rabbit anti-human HER2 mAb (2165 S) from Cell Signaling.



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    Figure 3. Western blot analysis of RTK activation in A549-ACE2 cells after SARS-CoV-2 infection. Cell lysates from mock infection (Mock), A/PR8 (PR8), and SARS-CoV-2 infection at different time points were analyzed by Western blotting to detect active and total forms of EGFR (A), TrkA (B), and <t>HER2</t> (C). Protein bands were quantified using Sciugo software 2.0.1. The ratio of the phosphorylated form to total form was calculated and compared to the mock (set as 1.00).
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    Image Search Results


    LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent ERBB2 (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.

    Journal: Journal of the American Chemical Society

    Article Title: Hijacking Extracellular Targeted Protein Degrader–Drug Conjugates for Enhanced Drug Delivery

    doi: 10.1021/jacs.5c15047

    Figure Lengend Snippet: LIPTAC-mediated degradation of multiple extracellular proteins. (a,b) Schematic illustration of PD-L1 targeting LIPTAC, and Western blot showing total degradation of PD-L1 on MDA-MB-231 cells following 24 h treatment of 50 nM monomeric anti-PD-L1 parent, Atz, or LIPTAC containing Atz. Percent PD-L1 levels were quantified by ImageJ relative to PBS control. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. (c) Western blot analysis showing lysosome-dependent PD-L1 degradation on MDA-MB-231 cells. Cells were pretreated with either 500 nM Bafilomycin A (BafA) or 500 nM MG132 for 1 h followed by 24 h treatment with 50 nM LIPTAC. (d) Schematic illustration of HER2-targeting LIPTAC and Western blot analysis showing total HER2 degradation on MCF7 cells following 24 h treatment of monomeric Traz, LIPTAC and KineTAC. Percent ERBB2 (HER2) levels were quantified by ImageJ relative to PBS control. Data represent at least two independent experiments. (e) Schematic illustration of CXCR4-targeting LIPTAC and Western blot analysis showing total CXCR4 degradation on HeLa cells following 24 h treatment of Nb monomer, monomeric 142F1, or LIPTAC. (f,g) Schematic illustration of cleaved CDCP1 (cCDCP1)-targeting LIPTAC and degradation of CDCP1 in PL45 cells following 24 h treatment of 50 nM cCDCP1 binder CL03 IgG, full-length CDCP1 (flCDCP1) binder 4A06 IgG, or cCDCP1-specific LIPTAC. Each sample was tested in biological triplicate and error bars represent standard deviations. Statistics were calculated by unpaired two-tailed student t test. * P < 0.05. ** P < 0.01. ns, not significant.

    Article Snippet: Antibodies used included rabbit anti-human EGFR (Cell Signaling Technology, catalog no. 4267S, 1:1000), rabbit anti-human PD-L1 (Cell Signaling Technology, catalog no. 13684S, 1:1000), rabbit anti-human CXCR4 (Cell Signaling Technology, catalog no. 64837S, 1:1000), rabbit anti-human ERBB2 (Cell Signaling Technology, catalog no. 4290S, 1:1000), rabbit anti-human CDCP1 (Cell Signaling Technology, catalog no. 13794S, 1:1000), mouse anti-human α-tubulin (Cell Signaling Technology, 3873S, 1:3000), goat anti-human LDLR (R&D Systems, catalog no. AF2148, 1:1000), IRDye 800CW goat anti-rabbit IgG (LI-COR Biosciences, catalog no. 926-32211), IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences, catalog no. 926-68070, 1:5000), IRDye 800CW donkey anti-goat IgG (LI-COR Biosciences, catalog no. 926-32214, 1:5000), and peroxidase goat anti-rabbit IgG (H+L) (Jackson ImmunoResearch, catalog no. 111-035-144, 1:5000).

    Techniques: Western Blot, Control, Two Tailed Test

    Figure 3. Western blot analysis of RTK activation in A549-ACE2 cells after SARS-CoV-2 infection. Cell lysates from mock infection (Mock), A/PR8 (PR8), and SARS-CoV-2 infection at different time points were analyzed by Western blotting to detect active and total forms of EGFR (A), TrkA (B), and HER2 (C). Protein bands were quantified using Sciugo software 2.0.1. The ratio of the phosphorylated form to total form was calculated and compared to the mock (set as 1.00).

    Journal: Pathogens

    Article Title: Cellular Receptor Tyrosine Kinase Signaling Plays Important Roles in SARS-CoV-2 Infection

    doi: 10.3390/pathogens14040333

    Figure Lengend Snippet: Figure 3. Western blot analysis of RTK activation in A549-ACE2 cells after SARS-CoV-2 infection. Cell lysates from mock infection (Mock), A/PR8 (PR8), and SARS-CoV-2 infection at different time points were analyzed by Western blotting to detect active and total forms of EGFR (A), TrkA (B), and HER2 (C). Protein bands were quantified using Sciugo software 2.0.1. The ratio of the phosphorylated form to total form was calculated and compared to the mock (set as 1.00).

    Article Snippet: The following antibodies were used for immunoblotting: rabbit anti-human TrkA polyclonal antibody (Cell Signaling Technology, 2505S, Danvers, MA, USA), rabbit anti-human pTrkA monoclonal antibody (mAb) (Cell Signaling Tech, 4168S), rabbit anti-human HER2 mAb (Cell Signaling Tech, 2165S), rabbit anti-human pHER2 mAb (Cell Signaling Tech, 2243L), mouse anti-human EGFR mAb (Cell Signaling Tech, 2239S), rabbit anti-human pEGFR mAb (Cell Signaling Tech, 3777S), rabbit anti-human GAPDH mAb (Cell Signaling Tech, 2118S), anti-rabbit HRP conjugated (Bio Rad, 1706515, Hercules, CA, USA), and anti-mouse IgG HRP conjugated (R&D, HAF007).

    Techniques: Western Blot, Activation Assay, Infection, Software

    A. A flow diagram of the GigaAssay. Designed DNA mutations are transduced into a cell library. Cells are allowed to proliferate, then cells are assayed for HER2 phosphorylation. Cells are sorted into 4 bins using flow cytometry. DNA is extracted from the cells, and sequencing of the 4 bins is done. Bioinformatics and downstream analytics determine the functional activity of all mutants by quantifying the distribution of a mutant’s representation across the 4 bins. B. A cartoon representation of the fluorescent readout of pHER2 activity via immunostaining of pY1248 antibody as a surrogate for auto-activated phosphorylation of HER2. C. A flow diagram showing classification of mutants in the HER2 TK and JM domains. The left path denotes literature mutants while the right path has GigaAssay mutants. A single asterisk indicates data approximated from a literature review. Double asterisks indicate mutants which have one GOF call and one non-GOF call in technical replicates. Boxes colored green are for GOF mutations and light green is for mutants in which only one of two technical replicates were classified as GOF.

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: A. A flow diagram of the GigaAssay. Designed DNA mutations are transduced into a cell library. Cells are allowed to proliferate, then cells are assayed for HER2 phosphorylation. Cells are sorted into 4 bins using flow cytometry. DNA is extracted from the cells, and sequencing of the 4 bins is done. Bioinformatics and downstream analytics determine the functional activity of all mutants by quantifying the distribution of a mutant’s representation across the 4 bins. B. A cartoon representation of the fluorescent readout of pHER2 activity via immunostaining of pY1248 antibody as a surrogate for auto-activated phosphorylation of HER2. C. A flow diagram showing classification of mutants in the HER2 TK and JM domains. The left path denotes literature mutants while the right path has GigaAssay mutants. A single asterisk indicates data approximated from a literature review. Double asterisks indicate mutants which have one GOF call and one non-GOF call in technical replicates. Boxes colored green are for GOF mutations and light green is for mutants in which only one of two technical replicates were classified as GOF.

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Flow Cytometry, Sequencing, Functional Assay, Activity Assay, Immunostaining

    A. A histogram of activity scores of all mutants in the GigaAssay. Gray bars are activity scores at, or below that for WT. Green bars indicate scores that are greater than WT. Hatched bars have mutants with scores that could be classified as non-GOF or WT. Green stars indicate scores of the 10 benchmark GOF mutants. Red stars indicate the non-pY1248 GOF benchmark mutations. Black stars indicate activity scores for non-GOF benchmark mutations. B. A pie graph showing perecentage s of classified HER2 mutants in technical replicates 1 and 2 of the GigaAssay based on statistical tests. The green slice indicates the percentage of mutants with a GOF classification across both replicates; grey indicates the percentage of mutants with non-GOF classification across both replicates; light green indicates the percentage of mutants with one of two GOF classification call across replicates; an dark gray indicates the small percentage of mutants that lacked sufficient data in at least one replicate to produce a classificaiton.

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: A. A histogram of activity scores of all mutants in the GigaAssay. Gray bars are activity scores at, or below that for WT. Green bars indicate scores that are greater than WT. Hatched bars have mutants with scores that could be classified as non-GOF or WT. Green stars indicate scores of the 10 benchmark GOF mutants. Red stars indicate the non-pY1248 GOF benchmark mutations. Black stars indicate activity scores for non-GOF benchmark mutations. B. A pie graph showing perecentage s of classified HER2 mutants in technical replicates 1 and 2 of the GigaAssay based on statistical tests. The green slice indicates the percentage of mutants with a GOF classification across both replicates; grey indicates the percentage of mutants with non-GOF classification across both replicates; light green indicates the percentage of mutants with one of two GOF classification call across replicates; an dark gray indicates the small percentage of mutants that lacked sufficient data in at least one replicate to produce a classificaiton.

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Activity Assay

    Each possible single amino acid substitution is represented by an individual box in the grid. The alternate amino acid is represented on the Y-axis, while the position is represented on the X-axis. The color of the cell indicates the functional activity of the mutation as indicated by the color key. Black squares are the reference sequence, and gray boxes have no data. A. The shade of green in the boxes indicate the level of functional activity of the HER2 mutant. B. The color of the boxes indicates the classification of the mutants based upon statistical analysis. Data are from replicate 1. Corresponding figures for replicate 2 are in Supplementary Figs. 1 & 2 .

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: Each possible single amino acid substitution is represented by an individual box in the grid. The alternate amino acid is represented on the Y-axis, while the position is represented on the X-axis. The color of the cell indicates the functional activity of the mutation as indicated by the color key. Black squares are the reference sequence, and gray boxes have no data. A. The shade of green in the boxes indicate the level of functional activity of the HER2 mutant. B. The color of the boxes indicates the classification of the mutants based upon statistical analysis. Data are from replicate 1. Corresponding figures for replicate 2 are in Supplementary Figs. 1 & 2 .

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Functional Assay, Activity Assay, Mutagenesis, Sequencing

    Functional domains of the assayed regions (679-705, 708-882, 885-992) are depicted. Each column represents a residue position, while information known about that position are shown in the labeled rows. The number of GOF calls for all possible missense mutants at the position are shown at the top and known functional information about HER2 is shown below. Every 25 amino acids are numbered and have a bolded border.

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: Functional domains of the assayed regions (679-705, 708-882, 885-992) are depicted. Each column represents a residue position, while information known about that position are shown in the labeled rows. The number of GOF calls for all possible missense mutants at the position are shown at the top and known functional information about HER2 is shown below. Every 25 amino acids are numbered and have a bolded border.

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Functional Assay, Residue, Labeling

    All surface plots and ribbon diagrams are the WT TK domain HER2 structure (PDB: 3PP0) with one member of each pair rotated 180° about the Y axis: A. Position gradient colored as a rainbow. B. Published benchmark GOF positions (755, 769, 776, 777, 798, and 862). C. GOF positions identified in the GigaAssay, with benchmarks colored yellow, GOF variants requiring a 1 bp codon change are colored pale green, and new GOF positions in the TK domain identified by the GigaAssay colored dark green (726, 730, 733, 744, 755-757, 759, 769, 776-778, 783, 786, 790, 793, 799, 800, 801, 803, 812, 816, 818, 821, 839, 841, 860, 862, 869, 878, 879, 895, 897, 899, 921, 939, 956, 957, 962, 963, 965, 975, 988, 990, and 992). Mutants identified in the GigaAssay that only require a 1 bp codon change (726, 730, 733, 744, 755, 756, 757, 769, 776, 777, 778, 786, 790, 793, 799, 800, 812, 816, 819, 820, 839, 841, 842, 862, 879, 895, 897, 899, 939, 956, 957, 963, 965, 992). D. Distinct cluster of GOF mutations (Cluster 1: 939, 956, 957, 962, 963, 965: red; Cluster 2: 812, 818, 819, 820, 821, 988, 990, 992: yellow; Cluster 3: 839, 841, 869, 769, 842: blue; Cluster 4: 730, 755, 756, 757, 759, 793: orange). E. Positions with multiple substitutions (Multi-Sub) that are GOF variant positions (769, 755, 992, 776-G778, 783, 839). F. Patient GOF variant positions including benchmarks (733, 755, 776, 769, 777, 798, 816, 841). G. The catalytic loop (844-850): magenta) H. ATP binding site from PDB binding to PubChem ID:03Q SYR127063 ATP mimetic drug (726, 729, 734, 751, 753, 770, 774, 783, 785, 796, 800, 801, 804, 805, 850, 852, 862, 863, 864). I. Phosphorylation (aquamarine, positions 686, 733, 735, 759, 772, 779, 875, 877, 900, 923, 974, 977, 998) J. Two allosteric sites in each monomer [yellow (705-718) and light purple (761-765,790-792)] that shift upon the activation dimer. K. Heterodimerization with EGFR (706-712, 714, 716, and 718, 761, 764, 765, 768, 772, 790, and another site - 791, 936, 937, 938, 939, 940, 943, 948, 949, 950, 952, 953, 956, 957, 960, 961, 964, 965, 985, 988, 992) . L. Acceptor interface for the JM domain (786,790,793,799).

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: All surface plots and ribbon diagrams are the WT TK domain HER2 structure (PDB: 3PP0) with one member of each pair rotated 180° about the Y axis: A. Position gradient colored as a rainbow. B. Published benchmark GOF positions (755, 769, 776, 777, 798, and 862). C. GOF positions identified in the GigaAssay, with benchmarks colored yellow, GOF variants requiring a 1 bp codon change are colored pale green, and new GOF positions in the TK domain identified by the GigaAssay colored dark green (726, 730, 733, 744, 755-757, 759, 769, 776-778, 783, 786, 790, 793, 799, 800, 801, 803, 812, 816, 818, 821, 839, 841, 860, 862, 869, 878, 879, 895, 897, 899, 921, 939, 956, 957, 962, 963, 965, 975, 988, 990, and 992). Mutants identified in the GigaAssay that only require a 1 bp codon change (726, 730, 733, 744, 755, 756, 757, 769, 776, 777, 778, 786, 790, 793, 799, 800, 812, 816, 819, 820, 839, 841, 842, 862, 879, 895, 897, 899, 939, 956, 957, 963, 965, 992). D. Distinct cluster of GOF mutations (Cluster 1: 939, 956, 957, 962, 963, 965: red; Cluster 2: 812, 818, 819, 820, 821, 988, 990, 992: yellow; Cluster 3: 839, 841, 869, 769, 842: blue; Cluster 4: 730, 755, 756, 757, 759, 793: orange). E. Positions with multiple substitutions (Multi-Sub) that are GOF variant positions (769, 755, 992, 776-G778, 783, 839). F. Patient GOF variant positions including benchmarks (733, 755, 776, 769, 777, 798, 816, 841). G. The catalytic loop (844-850): magenta) H. ATP binding site from PDB binding to PubChem ID:03Q SYR127063 ATP mimetic drug (726, 729, 734, 751, 753, 770, 774, 783, 785, 796, 800, 801, 804, 805, 850, 852, 862, 863, 864). I. Phosphorylation (aquamarine, positions 686, 733, 735, 759, 772, 779, 875, 877, 900, 923, 974, 977, 998) J. Two allosteric sites in each monomer [yellow (705-718) and light purple (761-765,790-792)] that shift upon the activation dimer. K. Heterodimerization with EGFR (706-712, 714, 716, and 718, 761, 764, 765, 768, 772, 790, and another site - 791, 936, 937, 938, 939, 940, 943, 948, 949, 950, 952, 953, 956, 957, 960, 961, 964, 965, 985, 988, 992) . L. Acceptor interface for the JM domain (786,790,793,799).

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Variant Assay, Binding Assay, Activation Assay

    All surface plots and ribbon diagrams are of the predicted WT HER2 structure of the JM domain with AlphaFold. One member of each 3D structure pair rotated 180° about the Y axis: A . Position gradient colored as a rainbow; B . Conserved autoinhibitory segment of the JM region in EGFR; C . Positions with GOF variants identified in the GigaAssay; D . Conserved residues with EGFR; E . Conserved residues with all HER family members; F . Heterodimerization positions of the JM region with EGFR; G. Positions that bind to TRAF4 in EGFR; H. Positions in HER2 that are phosphorylated (PhosphoSite); I. Conserved autoinhibitory residues that bind to the TK domain in the JM region in EGFR ; Benchmark mutation 679L is shown in cyan; J. Positions that bind to ARNO in EGFR; K. Positions that bind to Anionic lipids in EGFR; L. Positions with variants observed in cancer patients. A ubiquitylation site is at position 716 (not shown).

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: All surface plots and ribbon diagrams are of the predicted WT HER2 structure of the JM domain with AlphaFold. One member of each 3D structure pair rotated 180° about the Y axis: A . Position gradient colored as a rainbow; B . Conserved autoinhibitory segment of the JM region in EGFR; C . Positions with GOF variants identified in the GigaAssay; D . Conserved residues with EGFR; E . Conserved residues with all HER family members; F . Heterodimerization positions of the JM region with EGFR; G. Positions that bind to TRAF4 in EGFR; H. Positions in HER2 that are phosphorylated (PhosphoSite); I. Conserved autoinhibitory residues that bind to the TK domain in the JM region in EGFR ; Benchmark mutation 679L is shown in cyan; J. Positions that bind to ARNO in EGFR; K. Positions that bind to Anionic lipids in EGFR; L. Positions with variants observed in cancer patients. A ubiquitylation site is at position 716 (not shown).

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Mutagenesis

    Tumor mutant data was obtained from individual publications, NIH GCD, ClinVar, COSMIC, and The Cancer Genome Atlas. 109 mutants from these data sources were compared to various sets of mutants with GOF calls from the GigaAssay. A. For each category, number of mutants found amongst the tumor sources (green) and number of mutants with GOF calls from the GigaAssay (Blue). B, C. Kaplan Meier plots representing survival of patients with various categories of mutations. These cohorts contain variant information known prior to and after the GigaAssay experiments. B. Survival of various cohorts of cancer patients with different sets of genetic dispositions. The first four cohorts are groups defined by prior knowledge of HER2 mutant status and survival; the last group consists of patient samples containing at least one novel GigaAssay GOF mutant. C. Comparison of survival of patients with tumors with different sets of GOF benchmark mutations. GOF benchmarks with visual indication of hyperphosphorylation relative to the other benchmarks are separated into groups. The groups are defined by the experiment in which the hyperphosphorylation is observed. Patients with GigaAssay hyperphosphorylation GOF variants have poorer survival.

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: Tumor mutant data was obtained from individual publications, NIH GCD, ClinVar, COSMIC, and The Cancer Genome Atlas. 109 mutants from these data sources were compared to various sets of mutants with GOF calls from the GigaAssay. A. For each category, number of mutants found amongst the tumor sources (green) and number of mutants with GOF calls from the GigaAssay (Blue). B, C. Kaplan Meier plots representing survival of patients with various categories of mutations. These cohorts contain variant information known prior to and after the GigaAssay experiments. B. Survival of various cohorts of cancer patients with different sets of genetic dispositions. The first four cohorts are groups defined by prior knowledge of HER2 mutant status and survival; the last group consists of patient samples containing at least one novel GigaAssay GOF mutant. C. Comparison of survival of patients with tumors with different sets of GOF benchmark mutations. GOF benchmarks with visual indication of hyperphosphorylation relative to the other benchmarks are separated into groups. The groups are defined by the experiment in which the hyperphosphorylation is observed. Patients with GigaAssay hyperphosphorylation GOF variants have poorer survival.

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Mutagenesis, Variant Assay, Comparison

    Steps in the HER2 activation pathway are shown as a schematic representation, listing regions and previously characterized GOF mutations and newly identified GOF mutations for each step. The cluster location of each GOF is indicated (C1-C4). B, D, E . Some models have a smaller EGFR TK domain reflecting its position behind the HER2 TK domain.

    Journal: medRxiv

    Article Title: New Gain-of-Function Mutations Prioritize Mechanisms of HER2 Activation

    doi: 10.1101/2025.03.03.25323043

    Figure Lengend Snippet: Steps in the HER2 activation pathway are shown as a schematic representation, listing regions and previously characterized GOF mutations and newly identified GOF mutations for each step. The cluster location of each GOF is indicated (C1-C4). B, D, E . Some models have a smaller EGFR TK domain reflecting its position behind the HER2 TK domain.

    Article Snippet: [ ] Samples were immunostained with primary monoclonal antibodies raised against human HER2 (rabbit mAb, Cell Signaling) and/or pHER2(Y1248) (mouse mAb, Thermo Fisher Scientific) and secondary antibodies conjugated to Alexa Fluor 647 (for HER2 immunostaining) or Alexa Fluor 488 (for pHER2 immunostaining) for control experiments.

    Techniques: Activation Assay

    The optimization of the ratio of transposon plasmid and transposase plasmid for transfection of anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) The schematic representation of the anti-HER2/HER2-13 CAR structure. ( B ) Diagram of the construction and expansion of CAR-T cells. On day 0, the isolated PBMC were electroporated with PiggyBac transposase plasmid along with transposon DNA plasmids encoding the PB-HER2(13)-CAR-puro. On day 1, magnetic beads and IL-2, IL-7 and IL-21 were added for expansion for 3–5 days, then the cells were followed by removal of the magnetic beads and continued culture for approximately 18–21 days. ( C , D ) The representative flow cytometry graphs of CAR + expression in CAR-T conducted with three different ratios (μg:μg) of transposon plasmid and transposase plasmid. ( E ) The analytic results of the total cell proliferation, CD3 + CAR + , CD4 + CAR + , CD8 + CAR + , CD4 + CAR + /CD8 + CAR + and CD3 + CAR + cell numbers. The data was from two different donors

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The optimization of the ratio of transposon plasmid and transposase plasmid for transfection of anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) The schematic representation of the anti-HER2/HER2-13 CAR structure. ( B ) Diagram of the construction and expansion of CAR-T cells. On day 0, the isolated PBMC were electroporated with PiggyBac transposase plasmid along with transposon DNA plasmids encoding the PB-HER2(13)-CAR-puro. On day 1, magnetic beads and IL-2, IL-7 and IL-21 were added for expansion for 3–5 days, then the cells were followed by removal of the magnetic beads and continued culture for approximately 18–21 days. ( C , D ) The representative flow cytometry graphs of CAR + expression in CAR-T conducted with three different ratios (μg:μg) of transposon plasmid and transposase plasmid. ( E ) The analytic results of the total cell proliferation, CD3 + CAR + , CD4 + CAR + , CD8 + CAR + , CD4 + CAR + /CD8 + CAR + and CD3 + CAR + cell numbers. The data was from two different donors

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: Plasmid Preparation, Transfection, Isolation, Magnetic Beads, Flow Cytometry, Expressing

    The differences in in vitro expansion abilities, CD3 + CAR + , CD4 + CAR + /CD8 + CAR + expression between anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) The proliferation of CAR-T cells or NT cells which were transfected with empty vector (PB-puro). The arrow indicated the different time points for adding puromycin. The data was from three different donors. ( B ) On the 7th and 14th day, respectively, the representative images of CAR-T cells constructed with two sequences of scFv were taken at a magnification of 200× to compare their morphology. The scale bars in the pictures are 50 μm. ( C ) Representative flow cytometry photos of CD3 + CAR + , CD4 + CAR + and CD8 + CAR + expression in the CAR-T cells and NT cells. ( D – G ) The analytic results of CD3 + CAR + , CD4 + CAR + , CD8 + CAR + and CD4 + CAR + /CD8 + CAR + cells. The data was from five (or seven) different donors. The difference between the anti-HER2 group and anti-HER2-13 group was determined by Student t -test, * p < 0.05; ns (not significant, p ≥ 0.05), n = 5

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The differences in in vitro expansion abilities, CD3 + CAR + , CD4 + CAR + /CD8 + CAR + expression between anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) The proliferation of CAR-T cells or NT cells which were transfected with empty vector (PB-puro). The arrow indicated the different time points for adding puromycin. The data was from three different donors. ( B ) On the 7th and 14th day, respectively, the representative images of CAR-T cells constructed with two sequences of scFv were taken at a magnification of 200× to compare their morphology. The scale bars in the pictures are 50 μm. ( C ) Representative flow cytometry photos of CD3 + CAR + , CD4 + CAR + and CD8 + CAR + expression in the CAR-T cells and NT cells. ( D – G ) The analytic results of CD3 + CAR + , CD4 + CAR + , CD8 + CAR + and CD4 + CAR + /CD8 + CAR + cells. The data was from five (or seven) different donors. The difference between the anti-HER2 group and anti-HER2-13 group was determined by Student t -test, * p < 0.05; ns (not significant, p ≥ 0.05), n = 5

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: In Vitro, Expressing, Transfection, Plasmid Preparation, Construct, Flow Cytometry

    The differences in memory (CD44, CD62L) and characteristic markers of T cell exhaustion (PD-1, LAG-3, TIM-3) between anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) Representative flow cytometry photos of memory and exhaustion marker expression in anti-HER2 and anti-HER2-13 CAR-T cells. ( B ) The analytic results of memory and exhaustion marker expression in CAR-T cell (0: fresh state). The data was from three different donors. ( C , D ) The influence on the cell viability, memory (CD44, CD62L) and key exhaustion indicators (LAG-3, TIM-3, PD-1) of anti-HER2 and anti-HER2-13 CAR-T cells under cryopreservation (0: fresh state, 1: one month, 6: six months). The data was from two different donors. The disparity between the anti-HER2 group and the anti-HER2-13 group was assessed through a Student’s t -test, which yielded ns (showing non-significant differentials, p ≥ 0.05)

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The differences in memory (CD44, CD62L) and characteristic markers of T cell exhaustion (PD-1, LAG-3, TIM-3) between anti-HER2 and anti-HER2-13 CAR-T cells. ( A ) Representative flow cytometry photos of memory and exhaustion marker expression in anti-HER2 and anti-HER2-13 CAR-T cells. ( B ) The analytic results of memory and exhaustion marker expression in CAR-T cell (0: fresh state). The data was from three different donors. ( C , D ) The influence on the cell viability, memory (CD44, CD62L) and key exhaustion indicators (LAG-3, TIM-3, PD-1) of anti-HER2 and anti-HER2-13 CAR-T cells under cryopreservation (0: fresh state, 1: one month, 6: six months). The data was from two different donors. The disparity between the anti-HER2 group and the anti-HER2-13 group was assessed through a Student’s t -test, which yielded ns (showing non-significant differentials, p ≥ 0.05)

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: Flow Cytometry, Marker, Expressing

    The anti-tumor effects of CAR-T cells on different HER2-positive or HER2-negative cancer cell lines. ( A ) The protein expression levels of HER2 in different tumor cells of lung adenocarcinoma (SPC-A-1 HER2+ /SPC-A-1) and breast cancer (MDA-MB-231 HER2+/ MDA-MB-231 and SKBR-3/SKBR-3 shHER2) were determined by western blotting, and the expression of β-actin was measured to confirm equal protein levels. ( B – D ) Cytotoxic effects of CAR-T cells on the above cancer cells. Cell viability was measured by colorimetric CCK-8 assay after treatment with CAR-T cells or NT cells for 24 h. The differences among CAR-T groups and NT on SPC-A-1 HER2+ /MDA-MB-231 HER2+ /SKBR-3 cells were determined by two-way ANOVA and post hoc Tukey multiple comparison tests. & p < 0.05, && p < 0.01, &&& p < 0.001 anti-HER2-13 CAR-T group vs. NT group; $ p < 0.05, $$$ p < 0.001 anti-HER2 CAR-T group vs. NT group. The CAR-T groups on SPC-A-1 HER2+ /SPC-A-1, MDA-MB-231 HER2+ /MDA-MB-231 and SKBR-3/SKBR-3 shHER2 cells were determined by two-way ANOVA and post hoc Tukey multiple comparison tests. # p < 0.05 anti-HER2-13 CAR-T+SPC-A-1 HER2 vs. anti-HER2-13 CAR-T+SPC-A-1, and anti-HER2-13 CAR-T+MDA-MB-231 HER2 vs. anti-HER2-13 CAR-T+MDA-MB-231; #### p < 0.0001 indicates the statistical differences between anti-HER2-13 CAR-T+SKBR-3 and anti-HER2-13 CAR-T+SKBR-3 shHER2 group; **** p < 0.0001 indicates the statistical differences between anti-HER2 CAR-T+SKBR-3 and anti-HER2 CAR-T+SKBR-3 shHER2 group. The line plots display the mean value ± standard deviation (SD) from biological replicates (n = 3 wells per experiment)

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The anti-tumor effects of CAR-T cells on different HER2-positive or HER2-negative cancer cell lines. ( A ) The protein expression levels of HER2 in different tumor cells of lung adenocarcinoma (SPC-A-1 HER2+ /SPC-A-1) and breast cancer (MDA-MB-231 HER2+/ MDA-MB-231 and SKBR-3/SKBR-3 shHER2) were determined by western blotting, and the expression of β-actin was measured to confirm equal protein levels. ( B – D ) Cytotoxic effects of CAR-T cells on the above cancer cells. Cell viability was measured by colorimetric CCK-8 assay after treatment with CAR-T cells or NT cells for 24 h. The differences among CAR-T groups and NT on SPC-A-1 HER2+ /MDA-MB-231 HER2+ /SKBR-3 cells were determined by two-way ANOVA and post hoc Tukey multiple comparison tests. & p < 0.05, && p < 0.01, &&& p < 0.001 anti-HER2-13 CAR-T group vs. NT group; $ p < 0.05, $$$ p < 0.001 anti-HER2 CAR-T group vs. NT group. The CAR-T groups on SPC-A-1 HER2+ /SPC-A-1, MDA-MB-231 HER2+ /MDA-MB-231 and SKBR-3/SKBR-3 shHER2 cells were determined by two-way ANOVA and post hoc Tukey multiple comparison tests. # p < 0.05 anti-HER2-13 CAR-T+SPC-A-1 HER2 vs. anti-HER2-13 CAR-T+SPC-A-1, and anti-HER2-13 CAR-T+MDA-MB-231 HER2 vs. anti-HER2-13 CAR-T+MDA-MB-231; #### p < 0.0001 indicates the statistical differences between anti-HER2-13 CAR-T+SKBR-3 and anti-HER2-13 CAR-T+SKBR-3 shHER2 group; **** p < 0.0001 indicates the statistical differences between anti-HER2 CAR-T+SKBR-3 and anti-HER2 CAR-T+SKBR-3 shHER2 group. The line plots display the mean value ± standard deviation (SD) from biological replicates (n = 3 wells per experiment)

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: Expressing, Western Blot, CCK-8 Assay, Comparison, Standard Deviation

    The potential mechanisms involved in cytotoxicities of CAR-T cells against HER2 positive or negative lung adenocarcinoma and breast cancer cell lines. ( A – H ) Cytokines (IFN-γ, TNF-α, IL-10, IL-6, IL-2, GM-CSF) and effector molecule (perforin, granzyme B) production were quantified by ELISA following CAR-T cell co-culture with tumor targets at a 2:1 effector-to-target ratio. Compared with the NT control group, the statistical significance was assessed using two-way ANOVA followed by Dunnett’s post hoc test: * p < 0.05, ** p < 0.01, *** p < 0.001; the data were from two independent experiments with three wells each

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The potential mechanisms involved in cytotoxicities of CAR-T cells against HER2 positive or negative lung adenocarcinoma and breast cancer cell lines. ( A – H ) Cytokines (IFN-γ, TNF-α, IL-10, IL-6, IL-2, GM-CSF) and effector molecule (perforin, granzyme B) production were quantified by ELISA following CAR-T cell co-culture with tumor targets at a 2:1 effector-to-target ratio. Compared with the NT control group, the statistical significance was assessed using two-way ANOVA followed by Dunnett’s post hoc test: * p < 0.05, ** p < 0.01, *** p < 0.001; the data were from two independent experiments with three wells each

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Control

    The effects of anti-HER2, anti-HER2-13 CAR-T cells and trastuzumab on the MDA-MB-231 HER2+ xenograft mouse model. ( A ) The establishment and treatment pattern of the breast cancer MDA-MB-231 HER2+ mouse model. ( B ) The body weight of mice in each group was measured throughout the entire treatment period. ( C ) The representative photographic documentation of resected murine mammary tumors. ( D ) The tumor weight of dissected MDA-MB-231 HER2+ in mean ± SEM of 3–5 tumors. ### p < 0.001 anti-HER2 CAR-T+IL-2 group vs. anti-HER2-13 CAR-T+IL-2 group, *** p < 0.001, NT+IL-2 group vs. anti-HER2 CAR-T+IL-2 groups, by two-way ANOVA followed by post-hoc Tukey’s multiple comparison. * p < 0.05, NT+IL-2 group vs. anti-HER2-13 CAR-T+IL-2 group by Student t -test; n = 5 per group

    Journal: Oncology Research

    Article Title: Investigation on the Anti-Cancer Effects of HER2-Targeted CAR-T Cells Engineered Using the PiggyBac Transposon System

    doi: 10.32604/or.2025.065394

    Figure Lengend Snippet: The effects of anti-HER2, anti-HER2-13 CAR-T cells and trastuzumab on the MDA-MB-231 HER2+ xenograft mouse model. ( A ) The establishment and treatment pattern of the breast cancer MDA-MB-231 HER2+ mouse model. ( B ) The body weight of mice in each group was measured throughout the entire treatment period. ( C ) The representative photographic documentation of resected murine mammary tumors. ( D ) The tumor weight of dissected MDA-MB-231 HER2+ in mean ± SEM of 3–5 tumors. ### p < 0.001 anti-HER2 CAR-T+IL-2 group vs. anti-HER2-13 CAR-T+IL-2 group, *** p < 0.001, NT+IL-2 group vs. anti-HER2 CAR-T+IL-2 groups, by two-way ANOVA followed by post-hoc Tukey’s multiple comparison. * p < 0.05, NT+IL-2 group vs. anti-HER2-13 CAR-T+IL-2 group by Student t -test; n = 5 per group

    Article Snippet: They were then incubated overnight at 4°C with monoclonal rabbit anti-human HER2 antibody (Cell Signaling Technology, 4290S, Danvers, MA, USA, 1:1000) or anti-β-actin mAb (mouse origin) (Proteintech, 66009-1, Rosemont, IL, USA, 1:1000).

    Techniques: Comparison