Journal: bioRxiv

Article Title: RNF43 truncating mutations mediate a tumour suppressor-to-oncogene switch to drive niche-independent self-renewal in cancer

doi: 10.1101/748574

Figure Lengend Snippet: a, Volcano plot showing proteins enriched after streptavidin pull-down of biotin-treated HEK293 cells stably expressing dox-inducible RNF43-BirA*. Striped line demarcates the empirical 0.01 False discovery rate (FDR) cut off. Significantly enriched Wnt/β-catenin pathway components are highlighted in red. b, Western blot analysis of endogenous destruction complex components that co-precipitated with the indicated RNF43 cancer variants expressed in HEK293T cells. Oncogenic RNF43 truncations are indicated in red. c,d Confocal microscopy analysis of the subcellular localisation of Axin1-GFP (c) and endogenous CK1α (d) upon expression of WT or oncogenic RNF43 (R519X). Scale bars represent 10 μm. RNF43 is visualised by Flag-staining. Asterisks indicate RNF43-expressing cells. e, Wnt luciferase-reporter activity in HEK293T cells expressing non-mutated RNF43 R519X (SSLS) or the ALAA, DLDD and Δ486-89 mutants. Average luciferase reporter activities ±s.d. in n = 2 independent wells are shown. IP; immunoprecipitation, IB; immunoblot, WT; wild-type.

Article Snippet: The following primary antibodies were used for immunoblotting (IB), immunofluorescence (IF) or immunoprecipitation (IP): goat anti-Axin1 (R&D systems), goat anti-CK1ε (Santa Cruz), goat anti-CK1α (Santa Cruz), rabbit anti-GSK3β (Cell Signaling), mouse anti-β-catenin (BD Transduction laboratories), rabbit anti-APC (Santa Cruz), rabbit anti-FLAG (Sigma-Aldrich), rat anti-HA (Roche), rabbit anti-V5 (Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), mouse anti-V5 (Genscript), mouse anti-GFP (Roche), mouse anti-Actin (MP Biomedicals).

Techniques: Stable Transfection, Expressing, Western Blot, Confocal Microscopy, Staining, Luciferase, Activity Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: RNF43 truncating mutations mediate a tumour suppressor-to-oncogene switch to drive niche-independent self-renewal in cancer

doi: 10.1101/748574

Figure Lengend Snippet: a, Top: Schematic experimental set up of the clonogenic assay. Bottom: Bright-field microscopy images of WT, TP53 KO and onco- RNF43/TP53 KO human colon organoid lines at day 2 and 14. Scale bars represent 1000 μm. b, Relative outgrowth of WT, TP53 KO and onco- RNF43/TP53 KO organoid lines treated with the PORCN inhibitor C59 (1 μM) for 7 days. Graph shows the outgrowth of organoids at 5 days after splitting (day 14) relative to organoids at day 2. Error bars represent ±s.d. of the mean of n = 3 experiments. c, Model for mode of action RNF43 LOF and onco-RNF43 variants. (Left) RNF43 performs a bifunctional tumour suppressor role by (I) targeting Wnt receptors for endocytosis and lysosomal degradation, and (II) by transiently interacting with the destruction complex to reconstitute its activity in the cytosol and re-establish Wnt pathway inhibition. This second suppressor role involves CK1-mediated phosphorylation and an unknown molecular activity of the flexible cytosolic tail of RNF43. (Middle) Classical loss-of-function (LOF) mutations prevent RNF43 function at the plasma membrane, leading to Wnt receptor overexpression and, consequently, hypersensitivity of cancer cells to Wnt. (Right) Onco-RNF43 truncated variants are stably expressed at the plasma membrane and retain E3 ligase-dependent Wnt receptor downregulating activity. However, due to loss of the cytosolic tail, these mutants selectively trap CK1 and Axin1 at the plasma membrane, which prevents destruction complex assembly and drives uncontrolled β-catenin-mediated transcription of target genes. WT; wild-type, LOF; loss-of-function, β-cat; β-catenin.

Article Snippet: The following primary antibodies were used for immunoblotting (IB), immunofluorescence (IF) or immunoprecipitation (IP): goat anti-Axin1 (R&D systems), goat anti-CK1ε (Santa Cruz), goat anti-CK1α (Santa Cruz), rabbit anti-GSK3β (Cell Signaling), mouse anti-β-catenin (BD Transduction laboratories), rabbit anti-APC (Santa Cruz), rabbit anti-FLAG (Sigma-Aldrich), rat anti-HA (Roche), rabbit anti-V5 (Sigma-Aldrich), mouse anti-FLAG (M2; Sigma-Aldrich), mouse anti-V5 (Genscript), mouse anti-GFP (Roche), mouse anti-Actin (MP Biomedicals).

Techniques: Clonogenic Assay, Microscopy, Activity Assay, Inhibition, Membrane, Over Expression, Stable Transfection

Journal: PLoS ONE

Article Title: Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas

doi: 10.1371/journal.pone.0143150

Figure Lengend Snippet: The same pancreatic cancer tissues and their adjacent normal tissues as used in were analyzed. The tissue lysates containing the same amount of total proteins were subjected to immunoblotting with antibody against MAP1S or LC3, respectively. β-Actin served as another loading control.

Article Snippet: The membranes were then incubated with primary antibody against MAP1S, LC3 or β-Actin (SC-47778, Santa Cruz Biotechnology, Inc.), and protein bands were detected with enhanced chemiluminescence (ECL) based on horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse #172–1011 and rabbit #172–1019, Bio-Rad) with the ECL Western Blotting Detection Reagents.

Techniques: Western Blot, Control

Journal: PLoS ONE

Article Title: Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas

doi: 10.1371/journal.pone.0143150

Figure Lengend Snippet: A) A real-time RT-PCR analysis of the impact of TGFβ on the expression of MAP1S gene in pancreatic cancer cell line PANC-1 and Capan-2. The mRNA levels in untreated samples were set as 1. B) An immunoblotting analysis of the protein levels of MAP1S and LC3 in untreated or TGFβ-treated PANC-1 and Capan-2 cells in the absence (Ctrl) or presence of lysosomal inhibitor Bafilomycin A1 (BAF). Lysates with the same amount of total proteins were loaded in each lane and β-Actin served as another loading control. C,D) Plots of relative levels of MAP1S (C) or LC3-II (D) as shown as a representative in panel (B). The levels of MAP1S in untreated cells were set as 1. Results are the means ± S.D. of at least three repeats and the differences were compared using Student’s t test. **, P≤0.01; and *, P≤0.05. E) A fluorescent imaging analysis of the impact of TGFβ on PANC-1 cells transiently transfected with a plasmid for 48 hrs to express GFP–LC3 in the absence (Ctrl) or presence of TGFβ and/or BAF for 12 hrs. F) A quantification of GFP–LC3-labelled autophagosomes as shown in panel (E). The data were the average number of GFP-LC3 punctate foci ± S.D. for ten randomly selected images with size of 512 pixels×512 pixels for each that covers about 10 cells on average. The significance of differences was determined by Student’s t test. ***P≤0.001.

Article Snippet: The membranes were then incubated with primary antibody against MAP1S, LC3 or β-Actin (SC-47778, Santa Cruz Biotechnology, Inc.), and protein bands were detected with enhanced chemiluminescence (ECL) based on horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse #172–1011 and rabbit #172–1019, Bio-Rad) with the ECL Western Blotting Detection Reagents.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Control, Imaging, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: Transforming Growth Factor TGFβ Increases Levels of Microtubule-Associated Protein MAP1S and Autophagy Flux in Pancreatic Ductal Adenocarcinomas

doi: 10.1371/journal.pone.0143150

Figure Lengend Snippet: A,B) Immunoblotting analyses (A) and plots (B) of the impact of TGFβ on autophagy flux marker LC3-II in MEF cells developed from wild-type (MAP1S+/+) or MAP1S knockout mice (MAP1S-/-) in the absence (Ctrl) or presence of Bafilomycin A1 (BAF). C,D) Immunoblotting analyses (C) and plots (D) of the impact of TGFβ on autophagy flux marker LC3-II in MEF cells developed from wild type mice (MAP1S+/+) treated with mixture of random sequence siRNAs (MOCK) or MAP1S-specific siRNA (MAP1S) in the absence or presence of BAF. E,F) Immunoblotting analyses (E) and plots (F) of the impact of TGFβ on autophagy flux marker LC3-II in MEF cells developed from knockout mice (MAP1S-/-) transiently transfected with empty vector (HA) or plasmid carrying HA-MAP1S in the absence or presence of BAF. G,H) Immunoblotting analyses (G) and plots (H) of the impact of TGFβ on autophagy flux marker LC3-II in Capan-2 cells treated with mixture of random sequence siRNAs (MOCK) or MAP1S-specific siRNA (MAP1S) in the absence or presence of BAF. The same amounts of total proteins were loaded and β-Actin served as another control. The relative intensities of LC3-II in untreated wild type cells were set as 1. Data were the means ± S.D. of at least three repeats and the significance of differences was tested by Student’s t test. *, P≤0.05; **, P≤0.01; and unlabeled, not significant. I) A diagram showing the mechanism by which TGFβ suppresses tumorigenesis through MAP1S-mediated autophagy flux.

Article Snippet: The membranes were then incubated with primary antibody against MAP1S, LC3 or β-Actin (SC-47778, Santa Cruz Biotechnology, Inc.), and protein bands were detected with enhanced chemiluminescence (ECL) based on horseradish peroxidase (HRP)-conjugated secondary antibodies (mouse #172–1011 and rabbit #172–1019, Bio-Rad) with the ECL Western Blotting Detection Reagents.

Techniques: Western Blot, Marker, Knock-Out, Sequencing, Transfection, Plasmid Preparation, Control