Journal: bioRxiv

Article Title: Mechanistic Elucidation of the Antitumor Properties of a Novel Death Receptor 5 Activator

doi: 10.1101/700906

Figure Lengend Snippet: A. Kaplan-Meier plot showing that patients with tumors overexpressing both EGFR and MYC are associated with a significantly worse survival than are patients with tumors expressing low levels of both EGFR and MYC. Patients ranked based on the expression of EGFR and MYC were classified into four groups, named “low EGFR low MYC (N=359)”, “low EGFR high MYC (N=220)”, “high EGFR low MYC (N=225)”, and “high EGFR high MYC (N=372)”. Overall Survival (OS) was compared among these groups. The F test was used to compare the variance between groups ( P > 0.05, ns; P ≤ 0.05, *; P ≤ 0.01, **; P ≤ 0.001, ***). B. Micrographs showing the morphology of the indicated MCF10A stable cell lines after 24 h treatment with 5 µM tcyDTDO, 12.5 ng/ml TRAIL, tcyDTDO + TRAIL, tcyDTDO + TRAIL + 10 µM Q-VD-OPH, or 10 µM Lapatinib. C. Immunoblot analysis of stable MCF10A cell lines treated for 24 h as in B. D. Model for how DDAs activate TRAIL/DR5-induced cell death in an oncogene-dependent manner. In the context of EGFR or HER2 overexpression, DDAs elevate ER stress resulting in transcriptional upregulation of DR5. Through a second mechanism, DDAs alter DR5 disulfide bonding to promote DR5 protein stabilization, oligomerization, and activation of pro-apoptotic signaling. Cytotoxicity of DDAs and TRAIL is also potentiated in MYC overexpressing cells.

Article Snippet: Stable cell lines were constructed as follows: Retroviral vectors encoding EGFR (Plasmid 11011 ( )) and HER2 (Plasmid 40978 ( )) were purchased from Addgene (Cambridge, MA).

Techniques: Expressing, Stable Transfection, Western Blot, Over Expression, Activation Assay

Journal: Cancers

Article Title: Potent Antitumor Activity of Liposomal Irinotecan in an Organoid- and CRISPR-Cas9-Based Murine Model of Gallbladder Cancer

doi: 10.3390/cancers11121904

Figure Lengend Snippet: Mutant ERBB2 cooperates with loss of p53 and leads to papillary GBC in recipient mice. ( A ) Top: Schematic of human ERBB2, indicating the location of two point mutants (S310F and V777L). Bottom: retroviral vector used to transduce organoids, that had been treated with an sgp53-containing plasmid (px459) to induce loss of p53. ( B ) Immunofluorescence for ERBB2 (top) and phospho-ERBB2 (bottom) on organoids harboring the indicated genetic alterations. ( C ) Tumor volumes 36 days after s.c. implantation of the respective organoids into recipient mice. All mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids exhibited tumor development, whereas sgp53;empty vector- and sgp53;ERBB2 wildtype organoids did not give rise to tumors over a four-month observation period. There was no significant difference in the tumor burden of mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids ( p = 0.999). ( D ) Mice transplanted with sgp53;ERBB2 S310F - and sgp53;ERBB2 V777L organoids reached endpoint criteria with a median survival of 79.5 days and 58.5 days, respectively. ( E ) H&E and IHC for CK19 and EGFP on tumors generated with sgp53;ERBB2 S310 - and sgp53;ERBB2 V777L organoids.

Article Snippet: The pMSCV- ERBB2 -IRES- EGFP was a gift from Martine Roussel (Addgene, Watertown, MA, USA, plasmid #91888).

Techniques: Mutagenesis, Retroviral, Plasmid Preparation, Transduction, Immunofluorescence, Generated

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

Journal: Theranostics

Article Title: The S100A4 Protein Signals through the ErbB4 Receptor to Promote Neuronal Survival

doi: 10.7150/thno.22274

Figure Lengend Snippet: S100A4 interacts with ErbB receptors and protects neurons in vitro via ErbB2 and ErbB4. ( A, B ) Pharmacological blocker of ErbB (PD158780) does not affect the S100A4-induced neurite outgrowth ( A ), but abolishes the pro-survival effect of S100A4 in the H 2 O 2 -treated neurons ( B ). The ErbB inhibitor alone ( A , B , ■) has no effect. Ctl, neurite outgrowth from untreated controls; Inh, treatment with PD158780 (20 μM) alone. In ( A , B ), S100A4 is used at 20 μM, four independent experiments. ( B ) CTL, neuronal viability in untreated controls, ANOVA vs cultures treated with H 2 O 2 and S100A4 in the absence of inhibitor (A4). ( C ) Knockdown of ErbB2 reduces S100A4-induced neuroprotection (A4, 20 μM). For shRNA treatment, the survival of transfected neurons was normalized to untreated controls (100%). *, vs untreated; #, S100A4-untreated vs S100A4-treated cells, two-way ANOVA. Here and henceforth, results are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. ( D ) S100A4 interacts with ErbB receptors in solution. Immunoblot of the cross-linked Sulfo-SBED-S100A4 and Fc-fusion constructs of ErbB1, 2, 3, 4 or mouse immunoglobulin G (IgG) and cross-linked. Representative of two independent experiments. (ctl) Binding of sulfo-SBED-S100A4 to Protein A beads with no prey (negative control). Lower panel, loading controls. IgG, double band corresponds to partially reduced heavy and light IgG chains. ( E ) SPR binding of S100A4 to immobilized ErbB1, 2, 3 and 4, representative of two independent experiments. ( F, G ) Pharmacological blockers of EGFR PD153035 ( F ) and AG1478 ( G ) do not inhibit the pro-survival effect of S100A4 in neurons subjected to oxidative stress in vitro . The EGFR inhibitors alone ( F , G , ■) have no effect. ( H ) Inhibitory antibodies to EGFR or ErbB3 do not have a significant effect on the S100A4-induced survival in the H 2 O 2 -treated (□) or untreated (■, Ab) neurons, four to five independent experiments. (I) Inhibitory antibodies to ErbB4 (Ab) do not decrease neuroprotective effect of EGFR ligand amphiregulin (AR), but specifically block neuroprotection by the ErbB4 ligand neuregulin (NRG). ( J ) Inhibitory antibodies to ErbB4 abolish the pro-survival effect of S100A4 in the H 2 O 2 -treated neurons. Antibodies alone (■, Ab) have no effect. H-J , CTL, neuronal viability in untreated controls, ANOVA vs cultures treated with H 2 O 2 and S100A4 in the absence of antibodies (A4).

Article Snippet: For knockdown experiments, hippocampal neurons were prior to plating electroporated with 2 μg plasmid DNA encoding shRNA for rat ErbB2 or nontargeting shRNA (OriGene, Rockville, MD, USA), both cloned in pGFP plasmid.

Techniques: In Vitro, Knockdown, shRNA, Transfection, Western Blot, Construct, Binding Assay, Negative Control, Blocking Assay