Journal: Scientific reports

Article Title: Cell-free expression of functional receptor tyrosine kinases.

doi: 10.1038/srep12896

Figure Lengend Snippet: Figure 1. (a) Schematic of cell-free co-translation of ERBB2-NLP and EGFR-NLP. (b) ERBB2 was cell-free produced in the presence and absence of DMPC or with DMPC and co-expressed Δ 49A1. FluoroTect™ GreenLys (Promega) was added for visualizing newly synthesized ERBB2 protein. After 4 hours of expression, cell-free reactions were centrifuged at 14,000 rpm for 10 minutes. Small aliquots of sample before centrifuging (total, T), and the supernatant (soluble, S) and pellet (P) after centrifuging, were collected. All samples were loaded along with a cell-free reaction mixture only (-). Gel images were taken using Molecular Dynamics Typhoon 9410 Molecular Imager from GE Healthcare. Full-length versions of gel images are presented in Supplementary Figure 5. (c) The ERBB2-NLP complex shows greatly enhanced solubility as compared to protein only or protein DMPC vesicles.

Article Snippet: ELISA was carried out using DuoSet IC ERBB2 ELISA kit from R&D Systems.

Techniques: Produced, Synthesized, Expressing, Solubility

Journal: Scientific reports

Article Title: Cell-free expression of functional receptor tyrosine kinases.

doi: 10.1038/srep12896

Figure Lengend Snippet: Figure 2. (a) NLP associated ERBB2 is tyrosine phosphorylated. Cell-free expressions were set up with and without (-) ERBB2 plasmid. Samples were collected at 2 hr, 5 hr, 8 hr and overnight (18 hr), resolved by SDS- PAGE and western blotted with anti-phospho-tyrosine ERBB2 antibody pY1248 and anti-ERBB2 antibody Ab-3 after stripping. (b) The NLP associated ERBB2 is phosphorylated independent of protein expression. Cell-free expressed ERBB2 (in replicate) was treated with calf-intestinal alkaline phosphatase (CIP) to remove the phosphate group and then Ni purified. The purified ERBB2-NLPs were incubated with ATP, Mn2+, Mg2+ and buffer to allow for re-phosphorylation. Samples were resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine antibody 4G10 and anti-ERBB2 antibody Ab-3. (c) Binding of trastuzumab to cell-free ERBB2-NLPs and cellular ERBB2 extract were measured by ELISA as described. Cell-free ERBB2-NLPs or ERBB2 extracted from HEK293 cells over-expressing ERBB2 receptor were exposed to varying concentrations of trastuzumab (0–500 nM). The Kds for the trastuzumab binding to cell-free ERBB2-NLP and cellular ERBB2 extract were 4.4 nM and 2.7 nM, respectively. Each data point represents the average value of triplicate measurements. (d) NLP associated EGFR is phosphorylated, and the presence of EGF in the cell-free reaction increases the level of phosphorylation. EGFR-NLPs showed low level of phosphorylation during cell-free expression. Adding EGF, the natural ligand of EGFR, increases the phosphorylation. Cell-free expressions were set up with and without (-) EGFR plasmid, with and without EGF. After an 8 hr reaction, cell-free mixtures were resolved by SDS-PAGE and western blotted with anti-phospho-tyrosine EGFR antibody pY1110 and anti-EGFR. The white vertical line represents a splicing event of lanes from the same Western blot. Full-length versions of all western blots are presented in Supplementary Figure 6.

Article Snippet: ELISA was carried out using DuoSet IC ERBB2 ELISA kit from R&D Systems.

Techniques: Plasmid Preparation, SDS Page, Western Blot, Stripping Membranes, Expressing, Purification, Incubation, Phospho-proteomics, Binding Assay, Enzyme-linked Immunosorbent Assay

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 LipofectamineTM reagent.

Techniques: Expressing, Western Blot, Derivative Assay, Comparison, Quantitative Proteomics, 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 LipofectamineTM reagent.

Techniques: Quantitative Proteomics, Biomarker Discovery, 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 LipofectamineTM 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 LipofectamineTM reagent.

Techniques: Western Blot, Clone Assay, Isolation, Selection, Stable Transfection, Transfection, Control, shRNA, Flow Cytometry, Fluorescence, Quantitative Proteomics, Phospho-proteomics, Biomarker Discovery, 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 LipofectamineTM reagent.

Techniques: shRNA, Expressing