Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Binding affinities to human HER2 and CD3 at 37 °C by surface plasmon resonance. Data are K D ( n = 12 technical replicates of a single experiment for HER2-XPAT protein and HER2(1x-N); n = 8 for HER2(1x-C) and uTCE). Surface plasmon resonance sensorgrams for these data are provided in Supplementary Figs. – . b – d , In vitro tumor cytotoxicity of HER2-XPAT protein and its metabolites following a 48-h incubation with co-cultures of huPBMCs and the high HER2-expressing human tumor cell lines (1:1 effector–target ratio) SKOV3 ( b ), BT-474 ( c ) or the medium-low HER2-expressing MCF7 cell line ( d ). e , In vitro cytotoxicity of HER2-XPAT protein and its metabolites against BT-474 cells co-cultured with huPBMCs (1:1 effector–target ratio). f , Impact of the protease-cleavable linker on in vitro cytotoxicity versus BT-474 cells co-cultured with huPBMCs. g , h , CD69-positive T cells ( g ) and IL-2 secretion ( h ) following 72-h incubation of huPBMC/SKOV3 co-cultures with HER2-XPAT protein or its unmasked form (uTCE). i , Target-dependent T-cell activation with HER2-XPAT protein and its metabolites. CD3-expressing Jurkat reporter T cells were incubated with BT-474 cells at a 5:1 effector–target ratio for 6 h, followed by quantification of NFAT -induced luciferase activity and measured in relative luminescence units (RLUs). Mean data for n = 2 technical replicates within one single experiment ( b – i ). Extended Data Table provides a summary of EC 50 values for the different forms of the HER2-XPAT proteins in the cytotoxicity and reporter T-cell activation assays.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Binding Assay, SPR Assay, In Vitro, Incubation, Expressing, Cell Culture, Activation Assay, Luciferase, Activity Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: CD3 + Jurkat reporter T cells were incubated with or without BT-474 cells at a 5ː1 effector-target ratio for 6 hours, followed by quantification of NFAT -induced luciferase activity. The graph shows individual data points from a single representative experiment. There were n = 2 biological repeats at each concentration tested within the same experiment for each cell line. HER2, human epidermal growth factor receptor 2; RLU, relative luminescence unit; uTCE, unmasked T-cell engager; XPAT proteins, TCE fused to XTEN polypeptides.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Incubation, Luciferase, Activity Assay, Concentration Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) promoted by the i.v. administration of equimolar doses (every week (QW) for 3 weeks) of HER2-XPAT protein (2.1 mg kg −1 ) or uTCE to NOG mice bearing established (maximum tolerated volume (MTV) ~185 mm 3 ) BT-474 human tumors. The dependence of tumor-resident proteases for activity was demonstrated by the lack of significant TGI in mice treated with HER2-XPAT-NoClvSite. The average body weight of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. b ,TGI ± s.e.m. ( n = 8 mice for each concentration tested within one single experiment) in established human HT-55 xenografts (MTV ∼150 mm 3 ) following i.v. administration of HER2-XPAT protein (QW for 4 weeks) and HER2-uTCE (0.9 mg kg −1 three times a week (TIW) for 4 weeks). HER2-XPAT-NoClvSite (QW for 4 weeks) had no impact on tumor growth. The average body weight ± s.e.m. of the mice bearing tumor xenografts remained generally stable for the duration of the experiment following dosing with the HER2-XPAT proteins or the vehicle control. c , T-cell activation in BT-474 human tumor xenografts and peripheral blood evaluated by flow cytometry on day 18 following equimolar TIW i.v. dosing with HER2-XPAT protein and HER2-uTCE (day 40 following tumor inoculation). HER2-XPAT and HER2-uTCE induced robust and comparable activation of intratumoral CD4 + and CD8 + T cells, whereas no trends for T-cell activation were apparent in blood samples in which HER2 was not present. Statistical differences in TGI and T-cell activation for test compounds versus vehicle were assessed using mixed-effects multiple comparison analyses followed by Tukey’s post hoc test.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activity Assay, Control, Activation Assay, Flow Cytometry, Comparison

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein was used to track proteolytic cleavage in PDX tumor-bearing mice. b , A representative SDS–PAGE gel showing XPAT protein cleavage forms arising in a single PDX-bearing mouse, after imaging by LI-COR Biosciences. Four bands representing HER2-XPAT protein and its three unmasked forms are visible in the tumor sample (green channel) while the other (red channel) predominantly shows the XPAT protein form. c , Concentration of XPAT protein forms in tumors and healthy tissues. Results are the mean ± s.d. from 20 mice within one single experiment, with tumors consisting of ten different PDX (Supplementary Table ); mice were injected with fluorescent dye-labeled HER2-XPAT or EpCAM-XPAT protein. Note that tissues for which ≥14 samples were below the limit of quantification (BLQ) were reported as ‘BLQ.’ Tissues with averages calculated using some samples with BLQ readings are marked with an asterisk.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Labeling, SDS Page, Imaging, Concentration Assay, Injection

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: HER2-XPAT protein was administered via short i.v. infusions (∼1–3 h), whereas the unmasked counterpart, due to its short half-life, was administered via 48-h continuous i.v. infusion to provide prolonged systemic exposure. a , A dose-escalation study with a single dose of HER2-XPAT protein administered to each cynomolgus monkey. *At doses below 21 mg kg −1 , a HER2-XPAT prototype was used. b , Dose de-escalation study of uTCE in NHPs administered as a 48-h continuous infusion due to its short half-life. c , Plasma concentrations in NHPs following administration of a single i.v. dose of HER2-XPAT protein (42 mg kg −1 , n = 2 NHPs; 25 mg kg −1 , n = 4 NHPs; 6 mg kg −1 , n = 12 NHPs; 2 mg kg −1 , n = 12 NHPs) or a 48-h continuous infusion of uTCE (0.2 mg kg −1 d −1 , n = 1 NHP; 0.1 mg kg −1 d −1 , n = 1 NHP; 0.06 mg kg −1 d −1 , n = 1 NHP). Data represent mean plasma concentration (±s.d. when n > 3 NHP treated) for the NHPs treated at each dose within one single experiment. d , e , Activation of circulating CD4 + T cells ( d ) and CD8 + T cells ( e ) 24 h after drug administration (HER2-XPAT protein, n = 16 NHPs; HER2-uTCE, n = 5 NHPs). f – h , Highest plasma levels of TNF-α ( f ), IL-6 ( g ) and IFN-γ ( h ) measured 0–24 h following drug administration (HER2-XPAT protein, n = 23 NHPs; HER2-uTCE, n = 8 NHPs). Note that the normal ranges for cytokine levels in NHP are as follows: IL-6 0.3–1.2 pg ml −1 , TNF-α 1–10 pg ml −1 , IFN-γ 1.1–8.0 pg ml −1 (ref. ). Data in d – f represent a single sample from each NHP receiving the test material within each single experiment. TNF, tumor necrosis factor; IFN, interferon.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Concentration Assay, Activation Assay

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , Mean plasma concentrations (±s.d.) following a single i.v. administration of HER2-XPAT protein (single plasma samples from n = 6 NHPs) and HER2-XPAT-NoClvSite ( n = 4 NHPs) within one single experiment. b , Plasma concentrations of HER2-XPAT(1x−N) and HER2XPAT(1x−C) relative to the entire HER2-XPAT protein, following a single i.v. dose of HER2-XPAT protein (25 mg kg −1 , n = 4 NHPs; 42 mg kg −1 , n = 4 NHPs) within one single experiment. c , Quantification of cleavage products with a similar molecular weight to the uTCE generated following incubation of fluorescent-labeled HER2-XPAT protein in plasma from cancer patients ( n = 11), patients with inflammatory diseases ( n = 27), healthy donors ( n = 4) and NHPs (with ( n = 6) and without ( n = 4) drug-induced inflammation). The plasma incubation experiment represents a closed system with no clearance mechanisms and will therefore overestimate the accumulation of cleavage products occurring in vivo. Horizontal bars represent median values; dots represent individual observations. P values are based on unpaired t -tests.

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Molecular Weight, Generated, Incubation, Labeling, In Vivo

Journal: Nature Cancer

Article Title: Precision-activated T-cell engagers targeting HER2 or EGFR and CD3 mitigate on-target, off-tumor toxicity for immunotherapy in solid tumors

doi: 10.1038/s43018-023-00536-9

Figure Lengend Snippet: a , An XPAT protein comprises a TCE core with two scFvs, one targeting CD3 and the other, a TAA. Each scFv is masked by a protease-releasable XTEN mask, unstructured, hydrophilic polypeptides that act as modular, tunable masks, in addition to extending the half-life of the TCE. Each XTEN mask connects to the TCE core via a protease-cleavable linker, designed to be cleavable by any of eight proteases from three different classes (matrix metalloproteinases, serine proteases and cysteine proteases) involved in cancer progression . b , Predicted structure of HER2-XPAT protein visualized using AlphaFold2 v.2.0, a machine-learning-based computational method for predicting protein structures with reasonable accuracy . Colors indicate anti-HER2 domain, pale green; anti-CD3 domain, light orange; XTEN masks, blue; protease-cleavable linker, red; linkers, gray. The model represents a static picture showing a plausible conformation of the unstructured XTEN masks and the length of unstructured XTEN relative to the folded antibody domains in an XPAT protein. c , XPAT proteins are expected to remain largely intact in healthy tissues, where protease activity is well controlled by protease inhibitors. XPAT protein unmasking occurs in two steps via one of two potential paths to the fully unmasked state. The two requisite cleavage events can occur in either order and each sequence (either the top or bottom paths shown) is equally likely. In aggregate, both 1x-N and 1x-C partially unmasked forms will exist, depending on the cleavage path. Removal of both XTEN masks liberates the unmasked HER2-TCE (uTCE). d , XPAT proteins are designed to exploit the dysregulated protease activity present in tumors versus healthy tissues and expand the therapeutic index of TCEs through preferential unmasking in the TME. The active uTCE promotes the formation of immunologic synapses between tumor and T cells, resulting in potent cytotoxicity. Notably, the uTCE has a short half-life and should be rapidly cleared, thereby sparing healthy tissues when the uTCE diffuses away from the TME. By design, the molecular weight of the uTCE (∼59 kDa) is sufficiently small to allow rapid kidney filtration .

Article Snippet: A phycoerythrin (PE)-labeled detection antibody against HER2 (CD340 (erbB2/HER2) antibody, BioLegend, 324406) was prepared at different dilutions in fluorescence-activated cell sorter (FACS) buffer.

Techniques: Activity Assay, Sequencing, Molecular Weight, Filtration

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Analysis of the immunohistochemical pathological reports for HER2 of 13 CRC patients in Jiangsu Provincial Hospital. B Western blot analysis of HER2 expression in murine and human CRC cell lines. C Expression and localization of HER2 detected by immunofluorescence on MC38 HER2 , MC38 Ctrl , CT26 HER2 , and CT26 Ctrl cells. D The mRNA expression of HER2 was measured by qPCR. ** P < 0.01. E The expressions of HER2 on the cell membrane were detected using flow cytometry. F The expression and localization of HER2 in HCT116 and HT29 cells were determined by immunofluorescence. Scale bars, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunohistochemical staining, Western Blot, Expressing, Immunofluorescence, Membrane, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A In vitro growth inhibition assay for HCT116 cells treated with MMAE and Paclitaxel for 24 h. B In vitro growth inhibition assay for HCT116 and HT29 cells. C In vitro growth inhibition assay for MC38 HER2 and MC38 Ctrl cells treated with RC48 for 48 h. D In vitro growth inhibition assay for MC38 Ctrl and MC38 HER2 cells treated with MMAE for 24 h. E Flow cytometry analysis of cell cycle after treatment with different concentrations of RC48 for 48 h. F Clonogenic assay of HCT116, CT26 HER2 and MC38 HER2 treated with RC48. Data represent the mean ± SEM of three replicates. * P < 0.05, ** P < 0.01, versus as indicated. n.s. not significant.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: In Vitro, Growth Inhibition Assay, Flow Cytometry, Clonogenic Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A MC38 Ctrl cells were treated with different concentrations of Disitamab (0, 10, 30, 100 μg/ml) for 48 h, and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. B MC38 HER2 and MC38 Ctrl cells were treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h and the expression of p-TBK1, p-IRF3, TBK1, IRF3 protein was detected by immunoblotting. C HCT116 was treated with 100 μg/ml RC48 for 48 h and 30 μM CPT11 for 12 h. D The expressions of HER2 on the cell membrane were detected using flow cytometry. MFI Mean fluorescence intensity. E Co-immunoprecipitation of endogenous HER2 and STING in HCT116. F The expression and localization of HER2 in MC38 HER2 were detected by immunofluorescence. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 10 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Expressing, Western Blot, Membrane, Flow Cytometry, Fluorescence, Immunoprecipitation, Immunofluorescence

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A – E MC38 HER2 (1 × 10 6 ) cells were inoculated in the C57BL/6 mice. Drugs were given as described in Materials and Methods. B , C Tumor volume and body weight were measured every two days. D , E Solid tumors were separated and weighed after the mice were sacrificed. The data represent the mean ± SEM of 10 mice per group. F Paraffin sections of tumor tissues were analyzed by H&E staining, PCNA immunohistochemistry staining, and TUNEL fluorescent staining. G The survival of tumor-bearing mice. H – M Same experiment with CT26 HER2 in BALB/c mice. The data represent the mean ± SEM of 7 mice per group. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 50 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Staining, Immunohistochemistry, TUNEL Assay

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: A Immunofluorescence staining for p-TBK1 and p-IRF3 in tumor tissue in C57BL/6. B , C Immunohistochemical detection of IFN-β secretion in tumor tissue in BALB/c and C57BL/6. D LDH release assay was performed using Tils achieved from the syngeneic model of CT26 HER2 as described in material and methods. E , F Examination of CD3 + , CD8 + , IFN-γ secretion in the tumor by flow cytometry in BALB/c and C57BL/6. * P < 0.05, ** P < 0.01 versus as indicated. n.s. not significant. Scale bar, 100 μm.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Lactate Dehydrogenase Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: A HER2-targeting antibody-MMAE conjugate RC48 sensitizes immunotherapy in HER2-positive colon cancer by triggering the cGAS-STING pathway

doi: 10.1038/s41419-023-06073-8

Figure Lengend Snippet: RC48 exhibits the capability to bind to HER2 receptors and undergo internalization, subsequently releasing the toxin MMAE. MMAE inhibits tubulin, leading to the disruption of the cell cycle and resulting in the efficient elimination of tumor cells. Moreover, through its interaction with HER2, RC48 can alleviate the inhibition induced by internalized HER2 on the STING pathway and its downstream signaling. These combined mechanisms enable RC48 to effectively impede the proliferation of HER2-positive tumors while also enhancing their sensitivity to immunotherapy.

Article Snippet: Anti-HER2 antibody (ab134182) was from Abcam plc.

Techniques: Disruption, Inhibition

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

Techniques: CCK-8 Assay

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

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

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

Techniques: Knockdown, Activation Assay, Expressing, Transfection

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

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