Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

Techniques: CCK-8 Assay

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

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

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

Techniques: Knockdown, Activation Assay, Expressing, Transfection

Journal: Heliyon

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

doi: 10.1016/j.heliyon.2022.e10410

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

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

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

Journal: Nature Communications

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

doi: 10.1038/s41467-021-23271-0

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

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

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

Journal: Scientific Reports

Article Title: A multifunctional DNA nano-scorpion for highly efficient targeted delivery of mRNA therapeutics

doi: 10.1038/s41598-018-28542-3

Figure Lengend Snippet: Characterization of AptDzy-DNS induced translocation. Confocal microscopy images of SK-BR-3 cells after incubation with 0.5 μM Dzy-DNS without aptamer, HAptDzy-DNS, NAptDzy-DNS, and the mixture of HAptDzy-DNS and NApt-Dzy-DNS for 2 h. Scale bars: 20 μm. (Note: NApt, anti-nucleolin aptamer; HApt, anti-HER2 aptamer; Apt, aptamer).

Article Snippet: Phospho-HER2/ErbB2 Antibody Sampler Kit and β-Actin polyclonal antibody were obtained from Cell Signaling Technology (Shanghai, China).

Techniques: Translocation Assay, Confocal Microscopy, Incubation

Journal: Scientific Reports

Article Title: A multifunctional DNA nano-scorpion for highly efficient targeted delivery of mRNA therapeutics

doi: 10.1038/s41598-018-28542-3

Figure Lengend Snippet: Cell cytotoxicity assay and proliferation assay. ( A ) CCK-8 assay of cell viability for SK-BR-3 cells incubated with DNS at various concentration. ( B ) CCK-8 assay of cell viability for SK-BR-3 cells incubated with AptDzy-DNS at different hours. ( C ) RT-PCR characterization of HER2 mRNA expression for SK-BR-3 cells incubated without and with AptDzy-DNS. The data error bars indicate means ± SD (n = 5). * P < 0.05, ** P < 0.01 (two-tailed Student’s t-test), ( D ) Western blot characterization of HER2 expression for SK-BR-3 cells incubated with 0.25 (Lane 1), 0 (Lane 2), 0.5 (Lane 3), 0.25 (Lane 4), and 0.125 (Lane 5) μM AptDzy-DNS.

Article Snippet: Phospho-HER2/ErbB2 Antibody Sampler Kit and β-Actin polyclonal antibody were obtained from Cell Signaling Technology (Shanghai, China).

Techniques: Cytotoxicity Assay, Proliferation Assay, CCK-8 Assay, Incubation, Concentration Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Two Tailed Test, Western Blot