ihc cst 2165s ab 10692490 phospho her3 erbb3 tyr1289  (Cell Signaling Technology Inc)

Journal: Scientific reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Fig. 1. Pyrotinib downregulates HER2 protein levels and suppresses phosphorylation of HER2, PI3K/AKT, and RAS/MAPK signaling pathways in SK-BR-3 and JIMT-1 cells. (a–d) Expression levels of HER2 and its downstream proteins in the PI3K/AKT and RAS/MAPK signaling pathways were analyzed by western blotting. (a) Both cell types were treated with lapatinib (0.001, 0.01, 0.1, 1 µM) for 24 h. (b) Both cell types were treated with lapatinib (1 µM) at different time points. (c) Both cell types were treated with pyrotinib (0.001, 0.01, 0.1, 1 µM) for 24 h. (d) Both cell types were treated with pyrotinib (0.1 µM) at different time points. Results are representative of 3 independent replicates.

Article Snippet: After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight.

Techniques: Phospho-proteomics, Protein-Protein interactions, Expressing, Western Blot

Journal: Scientific reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Fig. 2. Pyrotinib promotes HER2 degradation via the ubiquitin–proteasome pathway. (a) HER2 mRNA expression in SK-BR-3 and JIMT-1 cells treated with pyrotinib (0.5µM) or lapatinib (1µM) as assessed using RT-qPCR. (b–d) SK-BR-3 and JIMT-1 cells were treated with the lysosomal inhibitor Baf-A1 (20nM) or proteasome inhibitors Velcade (0.5 µM) or MG-132 (10µM) for 0.5 h, with DMSO as the control, followed by the addition of pyrotinib (0.5µM) for 0, 2, and 4 h. (e–f) Cells were subjected to MG132 (10 µM) treatment for 0.5 h or DMSO as the control, followed by the addition of pyrotinib (0.5 µM) for 0, 2, and 4 h. HER2 was immunoprecipitated from the lysates, and the samples were analyzed by immunoblotting with an anti-ubiquitin, anti-HER2, and anti-HSP70 antibodies. GAPDH served as the loading control. Results are representative of 3 independent replicates.

Article Snippet: After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight.

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot

Journal: Scientific reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Fig. 3. Pyrotinib promotes HER2 internalization and T-DM1 endocytosis. (a) Cells were treated with pyrotinib (0.5µM) or lapatinib (1 µM) for 0, 2, and 4 h and processed for immunofluorescence experiments using anti-HER2 antibody (green). Nuclei were stained with DAPI (blue) (×1000), Scale bar = 10 μm. (b) After labeling T-DM1 with pHrodo Deep Red (pHro-do-T-DM1), the cells were exposed to pHrodo-T-DM1 (1 µg/ mL) alone or in combination with pyrotinib (0.1µM) for 0, 2, and 4 h. pHrodo-T-DM1 emits red fluorescent signals within the cellular interior. Nuclei were stained with DAPI (blue) (×600). The quantification of T-DM1 fluorescence intensity is now shown in Supplementary Figure S2. Scale bar = 10 μm. Results are representative of 3 independent replicates.

Article Snippet: After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight.

Techniques: Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Scientific reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer.

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Fig. 5. Pyrotinib enhances the antitumor effect of T-DM1 in vivo. (a) Images of JIMT-1 xenografts harvested after 21 days of treatment with T-DM1 (10 mg/kg) with or without pyrotinib (2 mg/kg) (n = 6). (b) Changes in tumor weight in the examined mice. (c) Changes in tumor volume in the examined mice. (d) Body weight changes in mice after treatments. (e) Representative images displaying HE and IHC staining of xenograft tumor tissues (×400), scale bars = 50 μm. The histogram shows the average absorbance of HER2. (f) The protein expression levels of HER2 and its downstream signaling pathways in tumor tissues of each group were analyzed by western blot. GAPDH served as the loading control. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant. Results are representative of 3 independent replicates.

Article Snippet: After being treated with pyrotinib and lapatinib, the cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a blocking solution and HER2 Rabbit mAb (Cell Signaling Technology) at 4 °C overnight.

Techniques: In Vivo, Immunohistochemistry, Expressing, Protein-Protein interactions, Western Blot, Control

Journal: Molecular oncology

Article Title: Molecular imaging predicts trastuzumab-deruxtecan (T-DXd) response in head and neck cancer xenograft models.

doi: 10.1002/1878-0261.70056

Figure Lengend Snippet: Fig. 1. Structure of different HER2 therapies. Trastuzumab-800 consists of an IRDye800 fluorophore conjugated to the HER2 targeting antibody Trastuzumab. Trastuzumab-Emtansine (T-DM1) consists of a microtubule inhibitor (emtansine) that binds to tubulin and is linked to Trastuzumab via a non-reducible thioether linker. Trastuzumab-Deruxtecan (T-DXd) consists of a topoisomerase I inhibitor (deruxtecan) that is linked to Trastuzumab via a tetrapeptide linker that can be cleaved selectively by enzymes. NIR, near infrared.

Article Snippet: Primary antibodies were: HER2 human samples (prediluted, PA0571; Leica BioSystems), HER2 xenografts (1 : 500 dilution, 4290; Cell Signaling, Danvers, MA, USA) and cH2AX (1 : 300 dilution, 05-636; Millipore).

Techniques:

Journal: Molecular oncology

Article Title: Molecular imaging predicts trastuzumab-deruxtecan (T-DXd) response in head and neck cancer xenograft models.

doi: 10.1002/1878-0261.70056

Figure Lengend Snippet: Fig. 2. Analysis of HER2 expression across head and neck cell lines and human tumor samples. (A) Flow cytometry analysis of HER2 expression (red) versus unstained control (gray) in 10 000–40 000 cells for each cell line. FaDu and UMSCC-47 cells expressed low HER2 expression, whereas UMSCC-1 cells had negligible HER2 expression. (B) UMSCC-1, FaDu, and UMSCC-47 HER2 IHC stain in their xenograft models. UMSCC-1 cell line exhibited no HER2 expression, whereas other cell lines showed low HER2 expression (IHC 1+) (n = 3 each), Scale bar = 100 lm. (C) HER2 expression in HNSCC human samples showed varying HER2 expression ranging from no (0) expression to moderate (2+) expression (n = 40), Scale bar = 100 lm. HNSCC, head and neck squamous cell carcinoma; IHC, immunohistochemistry.

Article Snippet: Primary antibodies were: HER2 human samples (prediluted, PA0571; Leica BioSystems), HER2 xenografts (1 : 500 dilution, 4290; Cell Signaling, Danvers, MA, USA) and cH2AX (1 : 300 dilution, 05-636; Millipore).

Techniques: Expressing, Flow Cytometry, Control, Staining, Immunohistochemistry

Journal: Molecular oncology

Article Title: Molecular imaging predicts trastuzumab-deruxtecan (T-DXd) response in head and neck cancer xenograft models.

doi: 10.1002/1878-0261.70056

Figure Lengend Snippet: Fig. 3. In vivo and ex vivo imaging of Trastuzumab-IRDye800 to identify HER2 negative vs. positive xenografts. (A) High magnification view of FaDu cells incubated with Trastuzumab-IRDye800 showing positive binding at 24 h post incubation (n = 3), Scale bar = 50 lm. (B) In vivo NIRF images of Trastuzumab-IRDye800 in FaDu (top) and UMSCC-1 (bottom) xenografts. Drug shows localization in FaDu xenograft but not in UMSCC-1 xenograft at 72 h. Fluorescent signal seen on flank opposite to tumor at 72 h represents liver. Red circle represents where tumor xenograft cells were injected. (C) Ex vivo NIRF images of resected organs in FaDu and UMSCC-1 xenografts at 72 h post Trastuzumab-IRDye800 injection. (D) Drug uptake in tumors was quantified as MFIs (SD) normalized to that of muscle, *P < 0.05 between tumors from FaDu and UMSCC-1 xenograft mice. P-values derived from an unpaired T-test. (E) Representative H&E (Scale bar = 200 lm) and NIRF (Scale bar = 100 lm) images for Trastuzumab-IRDye800 uptake co-related with ex vivo findings from FaDu xenografts in 5 lm sections (Top). Representative images from UMSCC-1 xenograft showed no fluorescent signal for Trastuzumab-IRDye800 (bottom). (B–E) FaDu n = 3, UMSCC-1 n = 2; H&E, hematoxylin and eosin stain; MFI, mean fluorescent intensity; NIRF, near infrared fluorescence; PanCK, pan cytokeratin.

Article Snippet: Primary antibodies were: HER2 human samples (prediluted, PA0571; Leica BioSystems), HER2 xenografts (1 : 500 dilution, 4290; Cell Signaling, Danvers, MA, USA) and cH2AX (1 : 300 dilution, 05-636; Millipore).

Techniques: In Vivo, Ex Vivo, Imaging, Incubation, Binding Assay, Injection, Derivative Assay, H&E Stain

Journal: Molecular oncology

Article Title: Molecular imaging predicts trastuzumab-deruxtecan (T-DXd) response in head and neck cancer xenograft models.

doi: 10.1002/1878-0261.70056

Figure Lengend Snippet: Fig. 5. Antitumor activity of T-DXd compared with T-DM1 and Trastuzumab in tumors with low HER2 level. (A) Antitumor activity of T-DXd compared with T-DM1 and Trastuzumab in FaDu xenografts. Each point represents the mean and SEM; P-values derived from a type II ANOVA test between all treatment cohorts, and the dotted line on the x-axis at day 14 represents the day that mice were sacrificed in all treatment groups other than T-DXd due to reaching humane end points. Each mouse was given one dose of 10 mgkg1 of their treatment cohort on day 0. (B) H&E, HER2, and y-H2AX IHC staining of 5 lm sections of FaDu xenografts treated with either T-DXd (top) or Trastuzumab (bottom) at day 14 (Scale bar = 200 lm). (C) Representative sections showing HER2 expression vs y-H2AX staining in FaDu tumors. Black arrows on the image on the left represent HER2-positive cells, whereas red arrows show HER2 negative cells. Black arrows on the image on the right show HER2-positive cells that are also positive for y-H2AX, whereas red arrows show HER2 negative cells that are positive for y-H2AX staining. Scale bar = 50 lm. (A–C) Control and T-DXd n = 5, T-DM1 n = 4, Trastuzumab n = 3. H&E, hematoxylin and eosin stain; IHC, immunohistochemistry.

Article Snippet: Primary antibodies were: HER2 human samples (prediluted, PA0571; Leica BioSystems), HER2 xenografts (1 : 500 dilution, 4290; Cell Signaling, Danvers, MA, USA) and cH2AX (1 : 300 dilution, 05-636; Millipore).

Techniques: Activity Assay, Derivative Assay, Immunohistochemistry, Expressing, Staining, Control, H&E Stain

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib downregulates HER2 protein levels and suppresses phosphorylation of HER2, PI3K/AKT, and RAS/MAPK signaling pathways in SK-BR-3 and JIMT-1 cells. ( a – d ) Expression levels of HER2 and its downstream proteins in the PI3K/AKT and RAS/MAPK signaling pathways were analyzed by western blotting. ( a ) Both cell types were treated with lapatinib (0.001, 0.01, 0.1, 1 µM) for 24 h. ( b ) Both cell types were treated with lapatinib (1 µM) at different time points. ( c ) Both cell types were treated with pyrotinib (0.001, 0.01, 0.1, 1 µM) for 24 h. ( d ) Both cell types were treated with pyrotinib (0.1 µM) at different time points. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Phospho-proteomics, Protein-Protein interactions, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib promotes HER2 degradation via the ubiquitin–proteasome pathway. ( a ) HER2 mRNA expression in SK-BR-3 and JIMT-1 cells treated with pyrotinib (0.5µM) or lapatinib (1µM) as assessed using RT-qPCR. ( b – d ) SK-BR-3 and JIMT-1 cells were treated with the lysosomal inhibitor Baf-A1 (20nM) or proteasome inhibitors Velcade (0.5 µM) or MG-132 (10µM) for 0.5 h, with DMSO as the control, followed by the addition of pyrotinib (0.5µM) for 0, 2, and 4 h. ( e – f ) Cells were subjected to MG132 (10 µM) treatment for 0.5 h or DMSO as the control, followed by the addition of pyrotinib (0.5 µM) for 0, 2, and 4 h. HER2 was immunoprecipitated from the lysates, and the samples were analyzed by immunoblotting with an anti-ubiquitin, anti-HER2, and anti-HSP70 antibodies. GAPDH served as the loading control. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Ubiquitin Proteomics, Expressing, Quantitative RT-PCR, Control, Immunoprecipitation, Western Blot

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib promotes HER2 internalization and T-DM1 endocytosis. ( a ) Cells were treated with pyrotinib (0.5µM) or lapatinib (1 µM) for 0, 2, and 4 h and processed for immunofluorescence experiments using anti-HER2 antibody (green). Nuclei were stained with DAPI (blue) (×1000), Scale bar = 10 μm. ( b ) After labeling T-DM1 with pHrodo Deep Red (pHro-do-T-DM1), the cells were exposed to pHrodo-T-DM1 (1 µg/mL) alone or in combination with pyrotinib (0.1µM) for 0, 2, and 4 h. pHrodo-T-DM1 emits red fluorescent signals within the cellular interior. Nuclei were stained with DAPI (blue) (×600). The quantification of T-DM1 fluorescence intensity is now shown in Supplementary Figure . Scale bar = 10 μm. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Immunofluorescence, Staining, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: Pyrotinib promotes the antitumor effect of T-DM1 by increasing drug endocytosis in HER2-positive breast cancer

doi: 10.1038/s41598-025-03678-1

Figure Lengend Snippet: Pyrotinib enhances the antitumor effect of T-DM1 in vivo. ( a ) Images of JIMT-1 xenografts harvested after 21 days of treatment with T-DM1 (10 mg/kg) with or without pyrotinib (2 mg/kg) ( n = 6). ( b ) Changes in tumor weight in the examined mice. ( c ) Changes in tumor volume in the examined mice. ( d ) Body weight changes in mice after treatments. ( e ) Representative images displaying HE and IHC staining of xenograft tumor tissues (×400), scale bars = 50 μm. The histogram shows the average absorbance of HER2. ( f ) The protein expression levels of HER2 and its downstream signaling pathways in tumor tissues of each group were analyzed by western blot. GAPDH served as the loading control. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns = not significant. Results are representative of 3 independent replicates.

Article Snippet: After blocking with 5% nonfat milk or 3% bovine serum albumin (BSA), the membranes were incubated at 4 °C overnight with the primary antibodies HER2 Rabbit mAb, Phospho-HER2 (Tyr1221/1222) Rabbit mAb, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Rabbit mAb, and Phospho-Akt (Ser473) Rabbit mAb, which were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: In Vivo, Immunohistochemistry, Expressing, Protein-Protein interactions, Western Blot, Control