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Cell Signaling Technology Inc phosphorylated p her2
$EBA downregulates <t>HER2,</t> p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and <t>p-HER2</t> <t>(Y1221/1222)</t> in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.$
Phosphorylated P Her2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 310 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/phosphorylated+p+her2/Phospho-HER2%2FErbB2+(Tyr1221%2F1222)+Rabbit+mAb/pmc12871574-33-46-49
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phosphorylated p her2 - by Bioz Stars, 2026-09

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1) Product Images from "Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer"

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2026.5751

$EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.$
Figure Legend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Techniques Used: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Figure Legend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Techniques Used: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Figure Legend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Techniques Used: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

Membrane:

Article Title: Activity and mechanism of acquired resistance to tarloxotinib in HER2 mutant lung cancer: an in vitro study
Article Snippet: Total cell pellets were dissolved in lysis buffer, and a total of 15–30 μg of protein was loaded into SuperSep TM Ace (Wako) and blotted. .. Protein was transferred to the membrane [Trans-Blot ® TurboTM Mini PVDF Transfer Pack (Bio-Rad, Hercules, CA)], and membranes were probed using one of following antibodies: anti-HER2 (Cell Signaling Technology Cat# 2242, RRID:AB_331015), phosphorylated (p)-HER2 (Tyr1221/1222, Cell Signaling Technology Cat# 2243, RRID:AB_490899), anti-HER3 (Cell Signaling Technology Cat# 4754, RRID:AB_10691324), p-HER3 (Tyr1289, Cell Signaling Technology Cat# 4791, RRID:AB_2099709), anti-Akt (Cell Signaling Technology Cat# 9272, RRID:AB_329827), p-Akt (Ser473, Cell Signaling Technology Cat# 9271, RRID:AB_329825), anti-Erk1/2 (p44/42 MAPK, Cell Signaling Technology Cat# 9102, RRID:AB_330744), p-Erk1/2 (p44/42 MAPK, Thr202/Tyr204, Cell Signaling Technology Cat# 9100, RRID:AB_330741), and β-actin (Cell Signaling Technology Cat# 4967, RRID:AB_330288) overnight at 4 °C. .. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Cell Signaling Technology Cat# 7074, RRID:AB_2099233) was used as the secondary antibody.

other:

Article Title: Synergistic effects of Nrf2 inhibitor, brusatol treatment in combination with lapatinib, a selective HER2 tyrosine kinase inhibitor in HER2-positive cancers
Article Snippet: 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 (Ser473) (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.).

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

Article Title: Nrf2 Inhibitor, Brusatol in Combination with Trastuzumab Exerts Synergistic Antitumor Activity in HER2-Positive Cancers by Inhibiting Nrf2/HO-1 and HER2-AKT/ERK1/2 Pathways
Article Snippet: All primary antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA) including the antibodies against Nrf2 (1 : 1,000; cat. no. 12721), HO-1 (1 : 1,000; cat. no. 43966), HER2 (1 : 1,000; cat. no. 2242), phosphorylated (p)-HER2 (1 : 1,000; cat. no. 2243), AKT (1 : 1,000; cat. no. 9272), p-AKT (1 : 1,000; cat. no. 4060), ERK1/2 (1 : 1,000; cat. no. 4695), p-ERK1/2 (1 : 1,000; cat. no. 8544), and β -actin (1 : 2,000; cat. no. 3700).

Image Search Results

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control

Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A) HER2 expression in human breast cancer cell lines was examined by western blot; (B) Dose-response curves in SK-BR-3, BT-474, HCC1569, and HCC1954 cells after 6 d of treatment with TRA; (C) Dose-response curves in SK-BR-3 and BT-474 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d treatment with PYR; (D−G) Colony formation assays in SK-BR-3 (D,E) and BT-474 cells (F,G) treated with vehicle (CTRL), TRA (10 µg/mL), PYR (100 nmol/L), or a combination treatment (TRA+PYR). Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (H,I) Migration (H) and invasion (I) of SK-BR-3 cells were evaluated by transwell assays without or with matrigel-coated inserts; (J−M) SK-BR-3 (J,L) and BT-474 (K,M) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S1. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ****, P<0.0001.

Article Snippet: The primary antibodies were as follows: phosphorylated (p) HER2 (Cell Signaling Technology, Danvers, MA, USA, Cat#2243S), HER2 (Abcam, Cambridge, UK, Cat#ab134182), pAKT (Cell Signaling Technology, Cat#4060S), AKT (Cell Signaling Technology, Cat#4691S), pERK1/2 (Cell Signaling Technology, Cat#4370S), ERK1/2 (Abcam, Cat#ab17942), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Proteintech, Wuhan, China, Cat#10494-1-AP).

Techniques: Migration, Expressing, Western Blot

Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-sensitive HER2+ breast cancer cells. (A,B) SK-BR-3 (A) and BT-474 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) SK-BR-3 (C) and BT-474 (D) cells were treated with PYR for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Techniques: Western Blot

Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib inhibits proliferation, migration, invasion, and HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A) Dose-response curves of HCC1569 and HCC1954 cells after 3 d of treatment with PYR. IC50 values were determined after 3 d of treatment with PYR; (B−E) Colony formation assays in HCC1569 (B,C) and HCC1954 (D,E) cells treated with CTRL, trastuzumab (TRA, 10 µg/mL), PYR (100 nmol/L), or TRA+PYR. Relative number of colonies (normalized to CTRL) was quantified using ImageJ; (F) Wound healing assay for HCC1954 cells treated with CTRL, TRA, PYR, and TRA+PYR. Images were taken at 0, 24, and 48 h; (G,H) Migration (G) and invasion (H) of HCC1954 cells were evaluated by transwell assays without or with matrigel-coated insert; (I−L) HCC1569 (I,K) and HCC1954 (J,L) cells were treated with increasing concentrations of PYR for 24 h or indicated times with PYR. The levels of indicated protein were assessed by western blot; (M,N) HCC1569 (M) and HCC1954 (N) cells were treated with TRA for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S2. TRA, trastuzumab; PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

Techniques: Migration, Wound Healing Assay, Western Blot

Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib inhibits HER2 downstream pathways in trastuzumab-resistant HER2+ breast cancer cells. (A,B) HCC1569 (A) and HCC1954 (B) cells were treated with increasing concentrations of PYR for 24 h; (C,D) HCC1569 (C) and HCC1954 (D) cells were treated with PYR for the indicated times; (E,F) HCC1569 (E) and HCC1954 (F) cells were treated with TRA for the indicated times. All the levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. PYR, pyrotinib; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01; ***, P<0.001.

Techniques: Western Blot

Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in inhibiting proliferation and HER2 downstream pathway in HER2+ breast cancer cells. (A−D) SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cell lines were treated with TRA, TRA+PYR, or TRA+ PER at the indicated concentrations for 3 d. Proliferation of breast cancer cells was analyzed and normalized to CTRL (%); (E−H) SK-BR-3 (E), BT-474 (F), HCC1569 (G), and HCC1954 (H) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown in Supplementary Figure S3. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. ***, P<0.001; ****, P<0.0001.

Techniques: Western Blot

Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in inhibiting HER2 downstream pathways in HER2+ breast cancer cells. SK-BR-3 (A), BT-474 (B), HCC1569 (C), and HCC1954 (D) cells were treated with CTRL, TRA (10 µg/mL) combined with PYR (100 nmol/L) or PER (10 µg/mL) for the indicated times. The levels of pHER2, HER2, pAKT, AKT, pERK, ERK, and GAPDH were assessed by western blot. The quantifications of blots are shown above. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

Techniques: Western Blot

Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Figure Lengend Snippet: Pyrotinib is superior to pertuzumab in suppressing tumor growth in a xenograft model. (A) HCC1954 xenograft mouse model was treated with CTRL, TRA, PYR, TRA+PYR, or TRA+PER for 24 d. The black arrow indicates the initial time of treatment. Tumor volume was measured twice a week, and data are presented as ; (B) After 24 d of treatment, mice were sacrificed, and tumors were dissected and photographed; (C) Body weight of mice was measured twice a week; data are presented as ; (D) IHC staining was performed for pHER2, pAKT, pERK, and Ki-67 in paraffin sections of HCC1954 xenograft tumors; (E) IHC scores were quantified and presented. n=6/group. TRA, trastuzumab; PYR, pyrotinib; PER, pertuzumab; HER2, human epidermal growth factor receptor 2. *, P<0.05; **, P<0.01.

Techniques: Immunohistochemistry

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Migration, Expressing, Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Migration, Wound Healing Assay, Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Western Blot

Journal: Chinese Journal of Cancer Research

Article Title: Pyrotinib is effective in both trastuzumab-sensitive and primary resistant HER2-positive breast tumors

doi: 10.21147/j.issn.1000-9604.2024.02.03

Techniques: Immunohistochemistry

Cell growth inhibition in vitro of HER2+ breast cancer cell lines treated with monoclonal anti-HER2 antibodies. Cells were plated in 24-well plates and treated with vehicle (DMSO), 10 μg/ml trastuzumab, or 10 μg/ml trastuzumab + 10 μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05, **p-value <0.01; ***p-value <0.001; ****p-value <0.0001; ns, not significant according to multiple t test with Bonferroni correction.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Cell growth inhibition in vitro of HER2+ breast cancer cell lines treated with monoclonal anti-HER2 antibodies. Cells were plated in 24-well plates and treated with vehicle (DMSO), 10 μg/ml trastuzumab, or 10 μg/ml trastuzumab + 10 μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05, **p-value <0.01; ***p-value <0.001; ****p-value <0.0001; ns, not significant according to multiple t test with Bonferroni correction.

Article Snippet: The following primary antibodies were used: Ab anti-phosphorylated (p-) HER2 (CST#2243, 1:1000), Ab anti-PTEN (CST #9559, 1:1000), Ab anti-AKT (CST#9272, 1:1000), Ab anti-p-AKT S473 (CST#9271, 1:1000), Ab anti-S6K, Ab anti-p-S6K T389 (CST#9205, 1:1000), Ab anti-beta-actin (CST#4970, 1:5000).

Techniques: Inhibition, In Vitro

Responses of HER2+ breast cancer cell lines to the alpha-specific PI3K inhibitor alpelisib. (A) , Cells were plated in 24-well plates and treated with increasing doses of alpelisib. Cell growth was assessed after 6 days. Data were analyzed by GraphPad Prism (version 9.0) to generate drug response curves and relative IC50 values using the log (inhibitor) versus response-variable slope model (bars, SEM) with normalization of data defining the biggest number in each dataset as 100% and the smallest number in the same dataset as 0%. (B) , Estimated IC50 values of alpelisib in HER2 + breast cancer cell lines. CI, confidence of interval.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Responses of HER2+ breast cancer cell lines to the alpha-specific PI3K inhibitor alpelisib. (A) , Cells were plated in 24-well plates and treated with increasing doses of alpelisib. Cell growth was assessed after 6 days. Data were analyzed by GraphPad Prism (version 9.0) to generate drug response curves and relative IC50 values using the log (inhibitor) versus response-variable slope model (bars, SEM) with normalization of data defining the biggest number in each dataset as 100% and the smallest number in the same dataset as 0%. (B) , Estimated IC50 values of alpelisib in HER2 + breast cancer cell lines. CI, confidence of interval.

Techniques:

The alpha-specific PI3K inhibitor alpelisib increases the efficacy of anti-HER2 therapy in HER2+ breast cancer cell lines. (A) , Cells were plated in 24-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab, 10μg/ml trastuzumab + 10μg/ml pertuzumab, 1μM alpelisib, 1μM alpelisib + 10μg/ml trastuzumab, 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05; ***p-value <0.001; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B) , Western blot analysis of total and phosphorylated proteins of the HER2/PI3K pathway signaling. Cells were plated in 6-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab (T), 10μg/ml trastuzumab + 10μg/ml pertuzumab (T+P), 1μM alpelisib (A), 1μM alpelisib + 10μg/ml trastuzumab (A+T), 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab (A+T+P).

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: The alpha-specific PI3K inhibitor alpelisib increases the efficacy of anti-HER2 therapy in HER2+ breast cancer cell lines. (A) , Cells were plated in 24-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab, 10μg/ml trastuzumab + 10μg/ml pertuzumab, 1μM alpelisib, 1μM alpelisib + 10μg/ml trastuzumab, 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab. Cell growth was assessed after 6 days. Data were normalized to vehicle. Data represent means ± SEM. *p-value <0.05; ***p-value <0.001; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B) , Western blot analysis of total and phosphorylated proteins of the HER2/PI3K pathway signaling. Cells were plated in 6-multiwell and treated with vehicle (DMSO), 10μg/ml trastuzumab (T), 10μg/ml trastuzumab + 10μg/ml pertuzumab (T+P), 1μM alpelisib (A), 1μM alpelisib + 10μg/ml trastuzumab (A+T), 1μM alpelisib + 10μg/ml trastuzumab + 10μg/ml pertuzumab (A+T+P).

Techniques: Western Blot

Pharmacological synergy between alpelisib and trastuzumab in HER2+/ PIK3CA mutant breast cancer cell lines. HCC1954 and KPL4 cells were seeded in 96 well plate treated with increasing concentrations of each drug alone or in combination (up to 10μM alpelisib and 100μg/ml trastuzumab) for 7 days. Combination indices were determined using the Chou-Talalay test by CompuSyn software. Numbers inside each box indicate the ratio of viable treated cells to untreated cells, from three independent experiments. Synergy is defined as combination index (CI)<1, an additive effect as CI=1, and antagonism effect as CI>1.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Pharmacological synergy between alpelisib and trastuzumab in HER2+/ PIK3CA mutant breast cancer cell lines. HCC1954 and KPL4 cells were seeded in 96 well plate treated with increasing concentrations of each drug alone or in combination (up to 10μM alpelisib and 100μg/ml trastuzumab) for 7 days. Combination indices were determined using the Chou-Talalay test by CompuSyn software. Numbers inside each box indicate the ratio of viable treated cells to untreated cells, from three independent experiments. Synergy is defined as combination index (CI)<1, an additive effect as CI=1, and antagonism effect as CI>1.

Techniques: Mutagenesis, Software

Venn diagram of significantly up-regulated genes in HER2+/ PIK3CA mutant alpelisib-resistant (AR) and alpelisib + trastuzumab-resistant (ATR) derivatives.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Venn diagram of significantly up-regulated genes in HER2+/ PIK3CA mutant alpelisib-resistant (AR) and alpelisib + trastuzumab-resistant (ATR) derivatives.

Techniques: Mutagenesis

Kaplan-Meier curves showing relapse-free survival (RFS) of HER2+ breast cancer patients. Log-rank test was used for comparing two groups with high (red) and low (black) expression of ARL4A , SNRPG , ODC1 , AKR1C1 , TRIM16L , AKR1C2 , GLB1L2 , and BDH1 genes. Number of patients at risk are indicated below each panel. HR, hazard ratio.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Kaplan-Meier curves showing relapse-free survival (RFS) of HER2+ breast cancer patients. Log-rank test was used for comparing two groups with high (red) and low (black) expression of ARL4A , SNRPG , ODC1 , AKR1C1 , TRIM16L , AKR1C2 , GLB1L2 , and BDH1 genes. Number of patients at risk are indicated below each panel. HR, hazard ratio.

Techniques: Expressing

Effect of AKR1C1 siRNA knockdown in parental and alpelisib-resistant HER2+/ PIK3CA mutant breast cancer cell lines. (A) , HCC1954 and KPL4 parental and alpelisib-resistant (AR) cell lines were transfected with siRNA targeting the AKR1C1 or siRNA control (si-Cnt). Culture medium was replaced the next day with regular medium or alpelisib-containing medium, and replaced again at 4 days. Cell growth was assessed at day 6. Data were normalized to si-Cnt transfection. Data represent means ± SEM. *p-value <0.05; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B) , Protein levels of AKR1C1 in HER2+/PIK3CA mutant breast cancer cell lines transfected with siRNA targeting siRNA control or against AKR1C1 were tested by Western blot.

Journal: Frontiers in Oncology

Article Title: The effect of the alpha-specific PI3K inhibitor alpelisib combined with anti-HER2 therapy in HER2+/PIK3CA mutant breast cancer

doi: 10.3389/fonc.2023.1108242

Figure Lengend Snippet: Effect of AKR1C1 siRNA knockdown in parental and alpelisib-resistant HER2+/ PIK3CA mutant breast cancer cell lines. (A) , HCC1954 and KPL4 parental and alpelisib-resistant (AR) cell lines were transfected with siRNA targeting the AKR1C1 or siRNA control (si-Cnt). Culture medium was replaced the next day with regular medium or alpelisib-containing medium, and replaced again at 4 days. Cell growth was assessed at day 6. Data were normalized to si-Cnt transfection. Data represent means ± SEM. *p-value <0.05; ****p-value <0.0001 according to multiple t test with Bonferroni correction. (B) , Protein levels of AKR1C1 in HER2+/PIK3CA mutant breast cancer cell lines transfected with siRNA targeting siRNA control or against AKR1C1 were tested by Western blot.

Techniques: Knockdown, Mutagenesis, Transfection, Control, Western Blot

Radiation treatment increased the levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) at low and high doses in mouse bladder tumor line-2 (MBT-2) cells in a time-dependent manner. MBT-2 cells were exposed to radiation doses ranging from 2.5–10 Gy, followed by preparation of cell lysates. Western blot analysis shows a time-dependent increase in phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2).

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo

doi: 10.12659/MSM.909865

Figure Lengend Snippet: Radiation treatment increased the levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) at low and high doses in mouse bladder tumor line-2 (MBT-2) cells in a time-dependent manner. MBT-2 cells were exposed to radiation doses ranging from 2.5–10 Gy, followed by preparation of cell lysates. Western blot analysis shows a time-dependent increase in phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2).

Article Snippet: The primary antibodies to EGFR, phosphorylated EGFR (p-EGFR), HER-2, and phosphorylated HER-2 (p-HER-2) were obtained from Abcam (Cambridge, MA, USA).

Techniques: Western Blot

Lapatinib treatment down-regulated the radiation-mediated levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) in mouse bladder tumor line-2 (MBT-2) cells in a dose-dependent manner. Pretreatment of mouse bladder tumor line-2 (MBT-2) cells with lapatinib (100 nM and 200 nM) for 24 h followed by radiation of varying doses range from 2.5–10 Gy. The cell lysates were obtained after 2 h. Western blots were performed for phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2). β-actin was used as loading control. Lapatinib treatment down-regulated the radiation-mediated levels of p-EGFR, p-AKT, p-HER2 in MBT-2 cells in a dose-dependent manner.

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo

doi: 10.12659/MSM.909865

Figure Lengend Snippet: Lapatinib treatment down-regulated the radiation-mediated levels of phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2) in mouse bladder tumor line-2 (MBT-2) cells in a dose-dependent manner. Pretreatment of mouse bladder tumor line-2 (MBT-2) cells with lapatinib (100 nM and 200 nM) for 24 h followed by radiation of varying doses range from 2.5–10 Gy. The cell lysates were obtained after 2 h. Western blots were performed for phosphorylated epidermal growth factor receptor (p-EGFR), phosphorylated AKT (p-AKT), and phosphorylated HER-2 (p-HER2). β-actin was used as loading control. Lapatinib treatment down-regulated the radiation-mediated levels of p-EGFR, p-AKT, p-HER2 in MBT-2 cells in a dose-dependent manner.

Article Snippet: The primary antibodies to EGFR, phosphorylated EGFR (p-EGFR), HER-2, and phosphorylated HER-2 (p-HER-2) were obtained from Abcam (Cambridge, MA, USA).

Techniques: Western Blot

Lapatinib treatment combined with radiation resulted in an increased tumor suppressive effect in the mouse bladder tumor line-2 (MBT-2) xenografts. ( A ) The mouse tumor xenograft model was created by injecting MBT-2 cells subcutaneously in C3H/HeN mice and were divided into four groups, treated with lapatinib (200 mg/kg), radiation (15 Gy), and a combination of both, and with vehicle (control). Values are shown as mean tumor volumes per group. ( B ) The mice with tumors were sacrificed on the 8 th day. Photomicrographs of sections of the tumor tissues, treated with radiation, lapatinib, and combined radiation and lapatinib were recorded ×200 magnification, following immunohistochemical staining with primary antibodies for HER-2 and EGFR ( A1–H1 ).

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Lapatinib, a Dual Inhibitor of Epidermal Growth Factor Receptor (EGFR) and HER-2, Enhances Radiosensitivity in Mouse Bladder Tumor Line-2 (MBT-2) Cells In Vitro and In Vivo

doi: 10.12659/MSM.909865

Figure Lengend Snippet: Lapatinib treatment combined with radiation resulted in an increased tumor suppressive effect in the mouse bladder tumor line-2 (MBT-2) xenografts. ( A ) The mouse tumor xenograft model was created by injecting MBT-2 cells subcutaneously in C3H/HeN mice and were divided into four groups, treated with lapatinib (200 mg/kg), radiation (15 Gy), and a combination of both, and with vehicle (control). Values are shown as mean tumor volumes per group. ( B ) The mice with tumors were sacrificed on the 8 th day. Photomicrographs of sections of the tumor tissues, treated with radiation, lapatinib, and combined radiation and lapatinib were recorded ×200 magnification, following immunohistochemical staining with primary antibodies for HER-2 and EGFR ( A1–H1 ).

Article Snippet: The primary antibodies to EGFR, phosphorylated EGFR (p-EGFR), HER-2, and phosphorylated HER-2 (p-HER-2) were obtained from Abcam (Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining

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