her2 blocking antibody  (Bio-Rad)

Journal: ACS Omega

Article Title: Nanolipoprotein-Mediated Her2 Protein Transfection Induces Malignant Transformation in Human Breast Acinar Cultures

doi: 10.1021/acsomega.1c03086

Figure Lengend Snippet: Her2 carried by NLPs causes malignant-like organization in nonmalignant cells. (A) Nonmalignant cells cultured in lrECM form growth arrested polarized acinar-like structure with the basal organization of α6 integrin (green) and lateral organization of β-catenin (red). (B) Malignant breast cancer cells form disorganized structures with no cell polarity, which fail to growth arrest, as shown by the presence of mitotic figures (white arrow). (C) Nonmalignant cells treated with Her2-NLPs form apolar masses, which fail to growth arrest. (D) In contrast, nonmalignant cells treated with empty NLPs organize normally. (E) Nonmalignant cells treated with Her2-NLPs and a Her2 dimerization inhibitory antibody organize normally. (F) Significantly more structures organize well in nonmalignant, empty-NLP-treated, inhibitory antibody only, and Her2-NLP+ inhibitory antibody conditions, whereas Her2-NLP-treated and malignant cells are less likely to organize into polarized, growth-arrested acini ( n = 5 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (G) Her2-NLPs show dose–response behavior, with fewer structures organizing well with increasing NLP dosage ( n = 3 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (H) Her2-NLP-treated structures are more likely to contain a mitotic figure than untreated or empty-NLP-treated cells ( n = 3 biological replicates, * indicates post hoc test significance of p < 0.05, ** indicates post hoc test of p < 0.001). (I) Increasing the doses of Her2-NLPs from 0.5 to 25 μg/mL causes increasing abnormalities in the acinar structure.

Article Snippet: Cells in lrECM were transferred to dishes, allowed to gel for 20 min at 37 °C, then covered with complete culture media ± 5 μg/mL Her2-NLPs or empty NLPs or Her2-NLPs plus a Her2 blocking antibody (clone 4D5, MCA6092, Biorad) at 26.67 μg/mL (which represented 1:1 molar ratio with Her2-NLPs).

Techniques: Cell Culture

Journal: ACS Omega

Article Title: Nanolipoprotein-Mediated Her2 Protein Transfection Induces Malignant Transformation in Human Breast Acinar Cultures

doi: 10.1021/acsomega.1c03086

Figure Lengend Snippet: Her2-NLPs induce gene expression changes in nonmalignant 3D cultures. (A) PCA plot for all sample sequences shows that principal component 1 separates biological replicates, and principal component 2 separates Her2-NLP treated from other conditions. (B) Genes common to multiple comparisons include BCRYN1 for all NLP-treated cells versus untreated samples; and CCL5, CUX2, EBI3, and TGM2 for Her2-NLP-treated samples versus either empty-NLP or untreated samples. (C) Ingenuity pathway analysis (IPA) network comparing Her2-NLPs to empty-NLP disks predicts relationships among several biological families and transcriptional regulators involved in cancer progression and metastasis, including (D) ERK, Akt, p38MAPK, and STAT3. Genes in red indicate hits found in the dataset. Orange coloring indicates predicted activation of biomolecules, and blue indicates predicted inhibition. Deeper color saturation indicates more confidence in predicted regulation. (E) Secondary tumor formation is the top activated disease network associated with Her2-NLP treatment compared to untreated samples. (F) Organ degeneration is predicted to be inhibited with Her2-NLP treatment compared to untreated samples. Genes in red indicate upregulated genes found in the dataset, whereas genes in purple indicate those that were downregulated in the dataset. Orange coloring indicates predicted activation, and blue indicates predicted inhibition of the biological phenotype. Log 2 fold change ±, p < 0.05.

Article Snippet: Cells in lrECM were transferred to dishes, allowed to gel for 20 min at 37 °C, then covered with complete culture media ± 5 μg/mL Her2-NLPs or empty NLPs or Her2-NLPs plus a Her2 blocking antibody (clone 4D5, MCA6092, Biorad) at 26.67 μg/mL (which represented 1:1 molar ratio with Her2-NLPs).

Techniques: Expressing, Activation Assay, Inhibition