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 co-immunoprecipitation experiments from cells, the above myc-KRAS G12V , myc-NRAS G12V , and myc-HRAS G12V cDNAs were cloned into pBabePuro (Addgene vector 1764) , creating plasmids pBabePuro-myc-KRAS G12V , -NRAS G12V , and -HRAS G12V . pLX304-PIP5K1A-V5 was provided by the Duke RNAi core .

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 co-immunoprecipitation experiments from cells, the above myc-KRAS G12V , myc-NRAS G12V , and myc-HRAS G12V cDNAs were cloned into pBabePuro (Addgene vector 1764) , creating plasmids pBabePuro-myc-KRAS G12V , -NRAS G12V , and -HRAS G12V . pLX304-PIP5K1A-V5 was provided by the Duke RNAi core .

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 co-immunoprecipitation experiments from cells, the above myc-KRAS G12V , myc-NRAS G12V , and myc-HRAS G12V cDNAs were cloned into pBabePuro (Addgene vector 1764) , creating plasmids pBabePuro-myc-KRAS G12V , -NRAS G12V , and -HRAS G12V . pLX304-PIP5K1A-V5 was provided by the Duke RNAi core .

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 co-immunoprecipitation experiments from cells, the above myc-KRAS G12V , myc-NRAS G12V , and myc-HRAS G12V cDNAs were cloned into pBabePuro (Addgene vector 1764) , creating plasmids pBabePuro-myc-KRAS G12V , -NRAS G12V , and -HRAS G12V . pLX304-PIP5K1A-V5 was provided by the Duke RNAi core .

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

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: Gene networks targeted by miR-130a & miR-145. a Identification of miRNA targeted genes from TargetScan mouse database, which was intersected with mRNA expression microarray data comparing tumor Gr-1 + CD11b + cells with those from healthy control mice. Seven targets were common for miR-130a and -145. b IPA analysis of gene networks targeted by miR-130a (purple), miR-145 (blue), or both (orange) involving TGFβ and IGF pathways. c Validation of the major pathway mediators comparing tumor-associated myeloid cells with those from healthy controls, qRT-PCR (left) and Western blot (right). d qRT-PCR (left) and Western blot (right) from Gr-1 + CD11b + cells ex vivo treated with miR-130a or -145 or control mimics. e Immunofluorescence images of TβRII (Green), IGF1R (red), and DAPI (blue) in Gr-1 + CD11b + cells from the spleen of 4T1 tumor-bearing mice. scale bar: 10 μm. f M1/M2 cytokine ratio post restorations of TβRII, IGF1R, and IRS1 in Gr-1 + CD11b + cells that overexpress miR-130a or miR-145. The ratio of M1/M2 cytokines was calculated by dividing each M1 cytokine (TNFα, IL-12, GM-CSF) to M2 cytokine (IL-10, IL-4) as described in Material and Methods. g–l metastasis reduction by IGF1R inhibitor NT157: g The number of metastatic nodules of 4T1 tumor-bearing Tgfbr2 MyeKO and WT mice treated with NT157 ( n = 7–10). h The number of metastatic nodules in 4T1 tumor-bearing mice that received IGF1R inhibitor NT157. i qRT-PCR of ARG1, TGFβ1, and IL-10 in Gr-1 + CD11b + cells from 4T1 tumor-bearing mice treated with NT157 ( n = 5). j The number of metastatic nodules from mice bearing E0771 tumor treated with NT157 ( n = 5–6). k qRT-PCR of ARG1, TGFβ1, and IL-10 in Gr-1 + CD11b + cells from E0771 tumor-bearing mice treated with NT157 ( n = 4). l The number of metastatic nodules of 4T1 tumor-bearing mice treated with NT157 with CD8α neutralizing antibody or IgG as control ( n = 10). Mice with similar tumor weight were used for metastatic nodule counts (Supplementary Fig. ). Data are presented as mean±SEM, and Student’s t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For preventing the mRNA degradation of TβRII, IGF1R and IRS1 in Gr-1 + CD11b + cells by miR-130a or miR-145, overexpression constructs without 3′-UTR were utilized including FerH-mouse Tgfbr2 (Protein Expression Laboratory in Frederick National Laboratory for Cancer Research), pReceiver-mouse Igf1r (GeneCopoeia), pBABE-mouse Irs1 (Addgene).

Techniques: Expressing, Microarray, Quantitative RT-PCR, Western Blot, Ex Vivo, Immunofluorescence

Journal: Nature Communications

Article Title: miR-130a and miR-145 reprogram Gr-1 + CD11b + myeloid cells and inhibit tumor metastasis through improved host immunity

doi: 10.1038/s41467-018-05023-9

Figure Lengend Snippet: miR-130a and miR-145 targeted genes and correlation with human cancer. a Kaplan–Meier survival curve (METABRICS data) for breast cancer patients with high & low levels of miR-130a (left), miR-145 (middle), and both miR-130a and 145 (right) in tumor tissues. Log-rank test was used to analyze the survival data. HR hazard ratio with its 95% confidence interval. b Fold change of miR-130a and miR-145 expression in human immature myeloid cells co-cultured with human breast cancer cell line MDA-MB231, miR-93 as a positive control. c qRT-PCR for fold changes of miR-130a (left) and miR-145 (right) in sorted human monocytic and granulocytic subsets from peripheral blood of healthy donors ( n = 8) or patients with advanced GI cancer ( n = 8). d Correlation of GRB10 levels in human peripheral blood mononuclear cells (PBMCs) (GSE20189) with human lung cancer stages. e Heat map showing a correlation of three key IGF pathway mediators (IGF1R, GRB10, and IRS1) with stages of human lung cancer (GSE20189). f Correlation of GRB10 and TβRII expression levels (key mediators of both IGF and TGFβ pathways) with stages of human lung cancers (GSE20189). Yellow and blue colors indicate increased and decreased expression, respectively. The data were plotted with patients divided into stage I/II vs III /IV groups. The percentage of patients with higher expression level was calculated for each group as indicated. The data are represented as mean ± SEM, and Student’s t test or χ 2 -square test in ( e , f ) was performed * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: For preventing the mRNA degradation of TβRII, IGF1R and IRS1 in Gr-1 + CD11b + cells by miR-130a or miR-145, overexpression constructs without 3′-UTR were utilized including FerH-mouse Tgfbr2 (Protein Expression Laboratory in Frederick National Laboratory for Cancer Research), pReceiver-mouse Igf1r (GeneCopoeia), pBABE-mouse Irs1 (Addgene).

Techniques: Expressing, Cell Culture, Positive Control, Quantitative RT-PCR