Journal: Nature Communications

Article Title: Cell surface protein aggregation triggers endocytosis to maintain plasma membrane proteostasis

doi: 10.1038/s41467-023-36496-y

Figure Lengend Snippet: BS4 induces HER2 autophosphorylation ( a, b ). SkBr3 cells ± dual EGFR/HER2 kinase inhibitor lapatinib (HER1/2i) were incubated with BS4 for 2.5 min as indicated. Samples were analysed by immunoblot for total HER2 and phospho-HER2 (pHER2) ( a ). SkBr3 cells were incubated with BS4-dylight650 (red) for 2.5 min, after fixation stained for phosphorylated tyrosine residues (p-Y, green) and analysed by confocal microscopy ( b ). Localisation of PIP3 to BS4-induced HER2 aggregates ( c ). SkBr3 cells were transfected with a vector expressing the PIP3 sensor PH-AKT-GFP (green) for 16 h treated or not with the PI3K inhibitor LY294002 (PI3Ki) followed by incubation with BS4-dylight650 (red) for 2.5 min. After fixation samples were analysed by confocal microscopy. BS4 uptake is reduced by HER2 kinase but not PI3-kinase inhibition ( d ). SkBr3 cells were treated with CytoD, lapatinib (HER1/2i) or LY294002 (PI3Ki) as indicated and incubated with BS4-dylight650 for 30 min. After fixation BS4 uptake was quantified by flow cytometry; means ± SD, n = 6 independent experiments, ns (non-significant) P = 0.252, **** P < 0.0001; one-way ANOVA with Dunnett’s multiple comparison test. BS4-induced ADE is independent of Ras ( e , f ). SkBr3 cells were transfected with a plasmid expressing a dominant-negative mutant (S17N) of Ras for 16 h followed by incubation with BS4-dylight650 for 30 min. After fixation surface-bound BS4 was counterstained and samples analysed by confocal microscopy. Subtraction of surface from total antibody signal yielded the endocytosed pool ( e ) with transfected cell outlined. Results are quantified in ( f ); dots represent measurements from individual cells, red lines indicate the median; n ≥ 50 cells from three independent experiments, ns (non-significant) P = 0.532; two-tailed unpaired Student’s t test. Localisation of VAV2 to BS4-induced HER2 aggregates ( g ). SkBr3 cells were incubated with BS4-dylight650 (red) for 2.5 min. After fixation, samples were stained for endogenous VAV2 and analysed by confocal microscopy. Scale bars: 10 µm ( b , e , g ), 3 µm ( c ). Source data are provided as a Source Data file.

Article Snippet: Antibodies recognising the HER2 cytoplasmic domain (2242), pHER2 (2241), HER3 (12708), the HA-tag (2367) and EGFR (4267) were from Cell Signaling Technology.

Techniques: Incubation, Western Blot, Staining, Confocal Microscopy, Transfection, Plasmid Preparation, Expressing, Inhibition, Flow Cytometry, Dominant Negative Mutation, Two Tailed Test

Journal: Cancer Cell

Article Title: A living biobank of patient-derived ductal carcinoma in situ mouse-intraductal xenografts identifies risk factors for invasive progression

doi: 10.1016/j.ccell.2023.04.002

Figure Lengend Snippet:

Article Snippet: The ERBB2-GFP lentivirus functionality was tested by transfecting HEK-293T cells (ATCC) and performing a Western Blot with a HER2/ErbB2 and pHER2/ErbB2 antibody (Cell signaling) blocked in milk powder.

Techniques: Labeling, Recombinant, Electron Microscopy, Plasmid Preparation, Blocking Assay, Software

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: ERBB signaling constitutes the core of KRAS dependency (A) PPI network of the 30 genes generated with STRING and visualized with Cytoscape. Colors indicate log 2 FC (nodes’ size indicates absolute log 2 FC) in gene expression between 20 most mt KRAS-dependent and 20 most mt KRAS-independent cell lines. (B) Results from GO enrichment analysis of the 30 genes using ShinyGO. Only terms with FDR<0.01 are plotted. Expression of genes from each category was averaged among cell lines with smallest and highest KDS30 scores. ‘Any (L)’ represents mt KRAS cancer cell lines with 10 lowest KDS30 scores. ‘Any (H)’ represents mt KRAS cancer cell lines with 10 highest KDS30 scores. ‘Panc (L)’ and ‘Panc (H)’ represent mt KRAS pancreatic cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. ‘Lung (L)’ and ‘Lung (H)’ represent mt KRAS lung cancer cell lines with 4 lowest and 4 highest KDS30 scores, respectively. (C and E) EGFR/ERBB2 (in blue), MEK (in red) and RAF (in green) inhibitors that were found to selectively kill high KDS30 over low KDS30 PAAD (in C) or LUAD (in E) cancer cells, considering max top 40 drugs. PRISM_1ry: primary PRISM Repurposing screen, PRISM_2ry: secondary PRISM Repurposing screen, CTRP: Cancer Therapeutics Response Portal v2. Y-axis: difference in mean FC or mean AUC, see main text. Only inhibitors of EGFR/ERBB2/MEK/RAF with p<0.05 are shown. (D and F) Results from drug sensitivity data analysis for mt KRAS pancreatic cancer cells included in the PRISM 2ry screen (D) and mt KRAS lung cancer cell lines included in the CTRP screen (F). Mean AUC of high KDS30 cells was subtracted from mean AUC of low KDS30 cells and plotted on X-axis. See main text for thresholds. Student’s t - test was applied to determine the statistical significance to observe the difference and plotted on Y-axis. MEK inhibitors are highlighted in red. EGFR/ERBB2 inhibitors are highlighted in blue. NERA: neratinib; SELU: selumetinib; TRAM: trametinib; PD31: PD-318088; AFAT: afatinib; GEFI: gefitinib; AZD8: AZD8931; DACO: dacomitinib; TUCA: tucatinib; CANE: canertinib; COBI: cobimetinib; AS70: AS-703026; OSIM: Osimertinib; ERLO: erlotinib . See also Figure S3 .

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Generated, Expressing

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet: mt KRAS addicted lung cancer lines are highly sensitive to the combination of EGFR/ERBB2 inhibitors with MEK inhibitors High KDS30 H358 and low KDS30 LU99 lung cancer cells were treated with vehicle control (Ctrl), an EGFR/ERBB2 inhibitor and a MEK inhibitor, either alone or in combination as indicated on each figure panel, for 48 h and processed for western blotting as described in . pEGFR, pErbB2, pERK: phosphorylated EGFR, ErbB2, and ERK, respectively. c-Casp3 and c-PARP: cleaved Casp3 and PARP, respectively. Actin: loading control. Single ∗ and double ∗∗ indicate gels each protein is blotted from. See also Figure S4 .

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Western Blot

Journal: iScience

Article Title: Novel mutant KRAS addiction signature predicts response to the combination of ERBB and MEK inhibitors in lung and pancreatic cancers

doi: 10.1016/j.isci.2023.106082

Figure Lengend Snippet:

Article Snippet: pHER2/ErbB2 (Tyr-1221/1222) , Cell Signaling Technology , catalog no. 2243S.

Techniques: Recombinant, Expressing, CRISPR, Software