expression plasmid encoding human wildtype erbb2  (Addgene inc)

Journal: Journal of Nanobiotechnology

Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

doi: 10.1186/s12951-024-02600-7

Figure Lengend Snippet: Assessment of HER2 expression in a series of breast cell lines. ( A ) List of the cell lines employed in this study ( B ) Western blot analysis of HER2 expression on extracts from MDA-MB-231, MDA-MB-468, MCF7, BT474 and SKBR3 cells derived from different BCs; MCF10A cells, derived from non-tumorigenic breast epithelium, were used for comparison. α-Tubulin was used to normalise the amounts of proteins loaded in each lane. The histogram reports HER2 total expression as Relative Units (R.U.) compared to MCF10A cells. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( C ) Western blot analysis of the soluble form of HER2 (sHER2) present in the concentrated media from the same cells as in B). The histogram shows the relative quantification, expressed as Relative Units (R.U.), after normalization to protein loading through Ponceau S staining of the filter. Statistical significance is considered when **** p < 0.0001 ( ANOVA ). ( D ) Flow cytometry analysis of HER2 cell surface exposure in the same cell lines as in A). The percentages of cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The plots also report the positions of the isotypes as black lines. The histogram shows the relative quantification, expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( ANOVA )

Article Snippet: Subsequently, the chosen vector, HER2 (ERBB2) Human shRNA Plasmid Kit (Locus ID 2064, OriGene code TG320342) and the corresponding negative control vector (scramble shRNA control) were transfected with the Lipofectamine™ reagent.

Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

doi: 10.1186/s12951-024-02600-7

Figure Lengend Snippet: Brightfield imaged and SERS intensity maps of a selected cell for the Raman peaks at 1080 and 1580 cm − 1 for the cell lines: ( A ) MCF10A ( B ) MDA-MB-468 ( C ) SKBR3. Scale bar = 10 μm. ( D ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines based on the SERS analysis

Article Snippet: Subsequently, the chosen vector, HER2 (ERBB2) Human shRNA Plasmid Kit (Locus ID 2064, OriGene code TG320342) and the corresponding negative control vector (scramble shRNA control) were transfected with the Lipofectamine™ reagent.

Techniques: Biomarker Assay, Membrane

Journal: Journal of Nanobiotechnology

Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

doi: 10.1186/s12951-024-02600-7

Figure Lengend Snippet: Raman classification of breast derived cell lines expressing different HER2 levels. ( A ) Median + IQR spectra of SKBR3 (lime), MCF10A (green) and MDA-MB-468 (blue), with PCA loading expressing high variance, black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) 3D score plot of PC2-4 scores demonstrating the classification achieved for SKBR3, MCF10A and MDA-MB-468 cells using 3 components. ( C ) Box-plot of the distribution for each cell line of PC4 scores. The asterisks represent Wilcoxon nonparametric test results with p < 0.0001 when ****. ( D - E ) LDA supervised classification with the separation achieved using the latent variables 1 and 2, and the misclassification rate, respectively

Article Snippet: Subsequently, the chosen vector, HER2 (ERBB2) Human shRNA Plasmid Kit (Locus ID 2064, OriGene code TG320342) and the corresponding negative control vector (scramble shRNA control) were transfected with the Lipofectamine™ reagent.

Techniques: Derivative Assay, Expressing

Journal: Journal of Nanobiotechnology

Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

doi: 10.1186/s12951-024-02600-7

Figure Lengend Snippet: HER2 assessment in scrambled- and HER2-silenced SKBR3 cells. ( A ) Western blot analysis of HER2 on extracts from single clones isolated upon selection of SKBR3 stably transfected with a scrambled control (SCR) or with an shRNA directed against HER2 (HER2 shRNA Clones 1–3). α-Tubulin was used as loading control for each sample. ( B ) Flow cytometry analysis of HER2 cell surface exposure in the scrambled transfected SKBR3 (SCR) or in clone 3 (Clone 3). The cell counts are reported on the y axis, while the Fluorescence Intensity on the x axis. The histogram shows the relative quantification expressed as Mean Fluorescence Intensity (MFI). Statistical significance is considered when **** p < 0.0001 ( t -test). ( C ) Western blot analysis of HER2, AKT and the phosphorylated form levels in extracts from SKBR3 transfected with a scrambled control and from the HER2-silenced clone 3. The first histogram illustrates the quantification of HER2 relative to α-Tubulin and the second the AKT phosphorylation level to total AKT compared to the SCR control. Statistical significance is considered when *** p < 0.001 and **** p < 0.0001 ( t -test). ( D ) Brightfield images and SERS intensity maps of a selected cell for the peaks at 1080 and 1580 cm − 1 for scrambled- and HER2-silenced SKBR3 cells. Scale bar = 10 μm. ( E ) Relative quantification of HER2 biomarker on cell membrane of the analysed cell lines

Article Snippet: Subsequently, the chosen vector, HER2 (ERBB2) Human shRNA Plasmid Kit (Locus ID 2064, OriGene code TG320342) and the corresponding negative control vector (scramble shRNA control) were transfected with the Lipofectamine™ reagent.

Techniques: Western Blot, Clone Assay, Isolation, Selection, Stable Transfection, Transfection, Control, shRNA, Flow Cytometry, Fluorescence, Biomarker Assay, Membrane

Journal: Journal of Nanobiotechnology

Article Title: Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines

doi: 10.1186/s12951-024-02600-7

Figure Lengend Snippet: Raman spectra of the scrambled- and HER2-silenced SKBR3 cells (clone 3). ( A ): Median IQR spectra of scrambled (lime) and HER2-shRNA SKBR3 (red) with PCA loadings expressing high variance, as black lines (y is offset for clarity). Vertical dash lines are single peaks of interest; cyan vertical bands are relevant biological bands in cell classification. ( B ) The box plot of distribution of PCA scores for PC4 for the scrambled SKBR3 and silenced counterpart is shown, with a Wilcoxon statistic. ( C ) The score plot of PC1-PC4, evidencing the best spatial separation among scrambled and silenced SKBR3, is shown

Article Snippet: Subsequently, the chosen vector, HER2 (ERBB2) Human shRNA Plasmid Kit (Locus ID 2064, OriGene code TG320342) and the corresponding negative control vector (scramble shRNA control) were transfected with the Lipofectamine™ reagent.

Techniques: shRNA, Expressing

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Exosomes derived from MDA-MB-231 cells stimulate RANKL-induced osteoclastogenesis in vitro . (A) Representative transmission electron microscopy images of exosomes derived from MDA-MB-231 cells. Scale bar, 500 nm (left panel) and 100 nm (right panel). (B) Nanoparticle tracking analysis of exosomes derived from MDA-MB-231 cells. (C) Western blot analysis of whole cell lysates (WC) and exosomes (EXOs) prepared from MDA-MB-231 cells. (D) BMMs were treated with exosomes derived from MDA-MB-231 cells in the presence of RANKL and M-CSF for 4 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (E) BMMs were treated with exosomes derived from MDA-MB-231 cells treated with vehicle (Con) or the pan RAS inhibitor, salirasib (10 µ M), in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and quantification of TRAP-positive multinucleated cells. ** P<0.01. (F) BMMs were treated with exosomes derived from MCF-7 cells transfected with control vector (Con) or K-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. *** P<0.001. (G) BMMs were treated with exosomes derived from T47D cells transfected with control vector (Con), K-RASV12, H-RASV12 , or N-RASV12 vector in the presence of RANKL and M-CSF for 6 days. Representative TRAP staining images and number of TRAP-positive multinucleated cells. * P<0.05 and *** P<0.001. (H) BMMs were treated with exosomes derived from control vector (Con) or HER2 vector-transfected T47D cells in the presence of RANKL and M-CSF for 6 days. * P<0.05 and **** P<0.0001. RANKL, receptor activator of nuclear factor-κB ligand; BMMs, bone marrow-derived macrophages; TRAP, tartrate-resistant acid phosphatase; M-CSF, macrophage colony-stimulating factor; EXOs, exosomes; ALIX, apoptosis-linked gene 2-interacting protein X; HSP, heat shock protein; TSG101, tumor susceptibility 101; GM130, Golgi matrix protein 130; HER2, human epidermal growth factor receptor 2.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Derivative Assay, In Vitro, Transmission Assay, Electron Microscopy, Western Blot, Staining, Transfection, Plasmid Preparation

Journal: International Journal of Molecular Medicine

Article Title: RAS-stimulated release of exosomal miR-494-3p promotes the osteolytic bone metastasis of breast cancer cells

doi: 10.3892/ijmm.2023.5287

Figure Lengend Snippet: Identification of osteoclastogenic miRNAs in exosomes induced by RAS activation. (A) Expression levels of 28 miRNAs in exosomes derived from MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. (B) The expression levels of 11 selected miRNAs in exosomes derived from control or KRASV12 vector-transfected MCF-7 cells were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (C) Cellular expression levels of 11 selected miRNAs in MCF-7 cells transfected with the control or K-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. (D) The expression levels of eight selected miRNAs in exosomes derived from MDA-MB-231 cells treated with the control or salirasib (10 µ M) were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05 and ** P<0.01. (E) The expression levels of seven selected miRNAs in exosomes derived from T47D cells transfected with the control vector (Con) or N-RASV12 vector were determined using RT-qPCR. The results were normalized to U6 snRNA. * P<0.05, ** P<0.01 and **** P<0.0001. (F) Representative images of TRAP-positive osteoclasts in BMMs transfected with the indicated miRNAs. BMMs transfected with the indicated miRNAs (20 nM each) were stimulated with RANKL and M-CSF for 4 days. NC, miRNA mimic negative control. ** P<0.01. (G) The expression levels of miR-494-3p, miR-1915-3p, miR-4508 and miR-6869-5p in sera derived from patients with HER2-positive breast cancer (n=19) and triple-negative breast cancer (n=15). The results were normalized to U6 snRNA. * P<0.05, *** P<0.001 and **** P<0.0001. RT-qPCR, reverse transcription-quantitative PCR; M-CSF, macrophage colony-stimulating factor; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer; BMMs, bone marrow-derived macrophages.

Article Snippet: The HER2 expression vector (cat. no. HG10004-UT) was purchased from Sino Biological.

Techniques: Activation Assay, Expressing, Derivative Assay, Quantitative RT-PCR, Plasmid Preparation, Transfection, Negative Control, Real-time Polymerase Chain Reaction

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Optogenetic clustering and membrane translocation of the BcLOV4 photoreceptor.

doi: 10.1073/pnas.2221615120

Figure Lengend Snippet: Fig. 3. BcLOV4 clustering at the membrane allows for modular activation of the entire ErbB receptor family. (A) The intracellular domains of ErbB1-4 were fused to the C-terminus of BcLOV-mCherry. (B) Membrane translocation of BcLOV-ErbB1-4 in response to blue light. (Scale bar, 20 μm.) See also Movie S3. (C and D) Erk activation dynamics downstream of BcLOV-ErbB1-4 in response to ON and OFF steps of blue light. See also SI Appendix, Fig. S4. Data represent mean ± SEM of two replicates, with each replicate representing the mean of ~500 to 2,400 cells. (E) Membrane ruffling (black arrows) downstream of stimulation of BcLOV- ErbB1-4, indicative of RTK stimulation. Ruffling is strongest for ErbB1 and ErbB2, less for ErbB4, and absent for ErbB3 activation. (Scale bar, 20 μm.) See also Movie S4. (F) Kinase activity suppresses BcLOV-EGFR membrane translocation. Translocation was observed under light stimulation of BcLOV-EGFR harboring a kinase-inactivating D813N mutation, in either the presence or absence of 1 µM EGFR inhibitor erlotinib (EGFRi). (Scale bar, 20 μm.) Quantification (Right) shows ratios of mean membrane and cytoplasmic fluorescence of 15 cells per condition. Significance was tested by one-way t tests between individual groups and was assessed by comparing P values to Bonferroni-corrected significance level of α/3. ****P < 0.00001, ***P < 0.0001.

Article Snippet: ErbB2 was sourced from MSCV- human Erbb2- IRES- GFP, which was a gift from Martine Roussel (Addgene plasmid # 91888; http://n2t.net/ addgene:91888; RRID:Addgene_91888).

Techniques: Membrane, Activation Assay, Translocation Assay, Activity Assay, Mutagenesis, Fluorescence