Journal: Nature Communications

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

doi: 10.1038/s41467-018-05692-6

Figure Lengend Snippet: BioID identifies RAS interactomes. a Immunoblot detection of myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms (top) and biotinylated proteins with HRP-streptavidin after affinity capture (AC) with streptavidin-conjugated beads (middle) in the absence (−) and presence (+) of biotin. Empty vector-transduced cells (bottom) serve as a negative control. n = 2. b Representative images indicated myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms, as visualized by fluorescence microscopy. Scale bar: 5 μM. n = 1. c Immunoblot detection of total (T-) and phosphorylated (P-) ERK and AKT and d Crystal Violet staining of cells stably expressing myc-tagged BirA* alone or fused to the indicated RAS G12V isoforms. n = 2. e Hierarchical clustering of the z -scores derived from degree that the 477 interactome proteins were labeled by the indicated myc-tagged BirA*-RAS G12V proteins in triplicate cultures. f Venn diagram of the number of interactome proteins identified by BioID by the indicated BirA*-RAS G12V proteins. Where indicated, β-actin serves as a loading control

Article Snippet: For BirA* proximity-labeling experiments, human KRAS G12V (4B splice variant), NRAS G12V , and HRAS G12V cDNAs were created by gene synthesis and cloned in frame 3′ of myc-BirA* in the plasmid pcDNA3.1mycBioID (Addgene vector 35,700) , creating plasmid pcDNA3.1-myc-BirA*-KRAS G12V , pcDNA3.1-myc-BirA*-NRAS G12V , and pcDNA3.1-myc-BirA*-HRAS G12V .

Techniques: Western Blot, Plasmid Preparation, Negative Control, Fluorescence, Microscopy, Staining, Stable Transfection, Expressing, Derivative Assay, Labeling, Control

Journal: Nature Communications

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

doi: 10.1038/s41467-018-05692-6

Figure Lengend Snippet: Loss-of-function analysis identifies critical components of the RAS interactomes. Waterfall plot of the sgRNA log 2 enrichment score of each of the 474 genes encoding interactome proteins targeted by Cas9/CRISPR-mediated loss-of-function (filled circle) in cells transformed by KRAS G12V (top), NRAS G12V (middle), or HRAS G12V (bottom). Enrichment scores for control ribosomal and BRAF genes, as well as scramble sgRNA, are shown for reference. Top 1% of positively and negatively enriched genes are labeled (black: enriched in cells transformed by multiple RAS isoforms; red: enriched in cells transformed by only one RAS isoform)

Article Snippet: For BirA* proximity-labeling experiments, human KRAS G12V (4B splice variant), NRAS G12V , and HRAS G12V cDNAs were created by gene synthesis and cloned in frame 3′ of myc-BirA* in the plasmid pcDNA3.1mycBioID (Addgene vector 35,700) , creating plasmid pcDNA3.1-myc-BirA*-KRAS G12V , pcDNA3.1-myc-BirA*-NRAS G12V , and pcDNA3.1-myc-BirA*-HRAS G12V .

Techniques: CRISPR, Transformation Assay, Control, Labeling

Journal: Nature Communications

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

doi: 10.1038/s41467-018-05692-6

Figure Lengend Snippet: PIP5K1A binds specifically to KRAS G12V . a Plots of proximity labeling by myc-tagged BirA* fused to (top), and sgRNA enrichment scores in cells transformed with (bottom), KRAS G12V (black triangle), NRAS G12V (orange square), or BirA*-HRAS G12V (blue circle) for the indicated 19 kinases. b , c Immunoblot detection of the indicated immunoprecipitated (IP) and co-immunoprecipitated (CO-IP) myc-RAS G12V and PIP5K1A-V5 proteins. n = 2. d Immunoblot detection of IP endogenous KRAS with CO-IP endogenous PIP5K1A from SW620 cells. n = 1. e Recombinant GST-PIP5K1A and His10-KRAS G12V co-elution (eluates 2–4: E2, E3, and E4) from a Ni-charged column, as detected by Coomassie staining (bottom) or immunoblot (top). INPUT, flow through (FT), and the final wash (W) confirm protein mixtures and retention of proteins on the column. n = 2. f , g Immunoblot detection of IP recombinant f His10-KRAS G12V or g His10-HRAS G12V with CO-IP recombinant GST-PIP5K1A. n = 2. h Schematic representation of myc-KRAS G12V unaltered, when the HVR is replaced with that of HRAS (HHVR), and when the indicated seven lysine residues in the HVR are mutated to alanine (7A). i , j Immunoblot detection of IP myc-KRAS G12V and either i myc-KRAS G12V -HHVR ( n = 2) or j myc-KRAS G12V -7A ( n = 1) CO-IP with PIP5K1A-V5. Where indicated, INPUT lysates serve to confirm appropriate expression of the indicated proteins, the absence (−) of PIP5K1A-V5 or RAS isoforms serve as a control for the specificity of the CO-IP (note that the α-V5 INPUT immunoblot in c was run on a separate gel processed at the same time), and IgG or no antibody serve as a negative control

Article Snippet: For BirA* proximity-labeling experiments, human KRAS G12V (4B splice variant), NRAS G12V , and HRAS G12V cDNAs were created by gene synthesis and cloned in frame 3′ of myc-BirA* in the plasmid pcDNA3.1mycBioID (Addgene vector 35,700) , creating plasmid pcDNA3.1-myc-BirA*-KRAS G12V , pcDNA3.1-myc-BirA*-NRAS G12V , and pcDNA3.1-myc-BirA*-HRAS G12V .

Techniques: Labeling, Transformation Assay, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Recombinant, Co-Elution Assay, Staining, Expressing, Control, Negative Control

Journal: Nature Communications

Article Title: Interrogating the protein interactomes of RAS isoforms identifies PIP5K1A as a KRAS-specific vulnerability

doi: 10.1038/s41467-018-05692-6

Figure Lengend Snippet: PIP5K1A mediates oncogenic KRAS signaling. a Representative Crystal Violet staining, b intensity of Crystal Violet staining (mean ± SE, triplicate samples, three experiments, normalized to vector control), and c immunoblot detection of phosphorylated (P-) ERK and AKT and PIP5K1A in cells expressing the indicated RAS G12V proteins in the absence (vector) or presence of up to three separate PIP5K1A sgRNAs. * p = 0.05, one-sided Student’s t test. n = 2. d Immunoblot detection of P-AKT in Capan-1 cells stably transduced with a vector encoding Cas9 and no sgRNA (−) or PIP5K1A_1 sgRNA (+) and another vector encoding no transgene (vector), wild-type PIP5K1A, or PIP5K1A KM (detected with an α-V5 antibody). n = 2. e GO classifications showing fold enrichment and significance ( p values, Fisher’s exact test) based on GO enrichment analysis of proteins biotinylated by BirA*-KRAS G12V in the absence or in the presence of PIP5K1A. Where indicated, total (T-) ERK and AKT as well as β-actin serve as loading controls

Article Snippet: For BirA* proximity-labeling experiments, human KRAS G12V (4B splice variant), NRAS G12V , and HRAS G12V cDNAs were created by gene synthesis and cloned in frame 3′ of myc-BirA* in the plasmid pcDNA3.1mycBioID (Addgene vector 35,700) , creating plasmid pcDNA3.1-myc-BirA*-KRAS G12V , pcDNA3.1-myc-BirA*-NRAS G12V , and pcDNA3.1-myc-BirA*-HRAS G12V .

Techniques: Staining, Plasmid Preparation, Control, Western Blot, Expressing, Stable Transfection, Transduction

Journal: Histology and histopathology

Article Title: Advances in pediatric rhabdomyosarcoma characterization and disease model development

doi:

Figure Lengend Snippet: Genetically modified mouse models for RMS

Article Snippet: Keller et al., 2004b Multifocal RMS Gorlin syndrome associated Ptch1 +/− back- ground with Tamoxifen inducible Cre mediated SmoM2 (mutant Smoothened causing constitutive Hedgehog activation) expression 100% multifocal rhabdomyosarcomas; tumors arise at 5 weeks following Tamoxifen administration or at 9 weeks due to sporadic SmoM2 expression Mao et al., 2006 Pleomorphic RMS Conditional KRasG12V knockin somatic mutation in p53 null or heterozygote null background 100% tumors in p53 −/− 10 weeks after Cre electroporation and 40% tumors in p53 +/− background at a slower rate Tsumura et al., 2006 Pleomorphic RMS KRasG12V expression in a) p53 +/fl , b)p53 fl/fl , c) p53R172H/fl , d) p53 R172H/+ mice controlled by AhCre a) KRasG12V p53 +/fl : 6% at 112 days; b) KRasG12V p53 fl/fl : 94% at 48 days; c) KRasG12V p53 R172H/fl :100% at 51 days; d) KRasG12V p53 R172H/+ : 88% at 62 days Doyle et al., 2010 Open in a separate window Genetically modified mouse models for RMS >70% of the alveolar RMS tumors are characterized by translocation events that lead to the generation of a potent transcriptional activator Pax/FOXO1 fusion protein.

Techniques: Genetically Modified, Expressing, Muscles, Transgenic Assay, Activation Assay, Mutagenesis, Knock-In, Electroporation