Journal: Genes & Development

Article Title: Regulated proteolysis of Trop2 drives epithelial hyperplasia and stem cell self-renewal via ?-catenin signaling

doi: 10.1101/gad.196451.112

Figure Lengend Snippet: Trop2 is cleaved by RIP. Trop2 cleavage mutants are deficient in inducing self-renewal in vitro. (A) PEB-Trop2-Myc tag cells were treated with either DMSO (vehicle), DAPT, or TAPI-2, followed by Western blot. The red arrow indicates accumulated ICP. The ICD cannot be detected without immunoprecipitation (IP). The blue arrow indicates full-length mTrop2 (FLTrop2). Full-length Trop2 appears between 35 and 50 kD. One out of three independent experiments is shown. Schematic representation of the generation of 15-kD ICP upon treatment with DAPT is shown on the right. (B) TRAMP-C2 expressing mTrop2 with a C-terminal Myc tag to follow the ICD were treated with DMSO (vehicle), DAPT, or TAPI-2. Twenty-four hours post-treatment, cells were fixed and stained with anti-Myc tag or anti-ECD antibody and DAPI and subjected to confocal microscopy. Bar, 10 μm. One out of two independent experiments is shown. The graph represents the percentage of cells with nuclear ICD. (C) PEB expressing Trop2-Myc tag cells were transfected with nontargeting (Cont), PS-1, PS-2, or both PS-1 and PS-2 siRNA, followed by Western blot with the indicated antibodies. Similar to DAPT treatment, knockdown of presenilins caused ICP appearance, as indicated by the red arrow. The blue arrow shows full-length Trop2 (FLTrop2). One out of three independent experiments is shown. (D) PEB cells were infected with lentivirus expressing RFP (control), mTrop2-Myc tag and RFP (mTrop2), or V286K-Myc tag (V286) mutant. Cells were lysed and subjected to Western blot with antibodies against Myc tag or the ECD of Trop2. The red arrow shows ICP appearance in V286K, demonstrating not fully processed Trop2. One out of three independent experiments is shown. (E) Serum-free medium from TRAMP-C2 cells transduced with RFP lentivirus (control), mTrop2-Myc tag (mTrop2), or V188K-Myc tag (V188K) was precipitated, followed by Western blot using commercial anti-ECD antibody or anti-Myc tag recognizing the ICD. Only the ECD of Trop2 was detected in the precipitated media from cells expressing wild-type Trop2, but not from cells expressing V188K. Two independent experiments were performed. (F) LSCThi cells were isolated by FACS, and an equal number of cells was infected with RFP (control), mTrop2, V286K, or V188K mutants expressing lentivirus and plated in triplicate. Sphere number and diameter in microns were quantified after Gen 1 and Gen 2. (Left graphs) Sphere number in each sample is presented as the percentage normalized to the RFP-infected spheres (control). Data are represented as mean ± SEM. Sphere size measured by sphere diameter in microns is presented in the right graphs. Purple bars represent RFP (control), blue bars indicate mTrop2, yellow bars represent V286K mutant, and green bars indicate V188K mutant-infected spheres. Each sample in the experiment was plated in triplicate. Three independent experiments were performed.

Article Snippet: Western blot analysis and immunoprecipitation experiments were performed using the following antibodies: anti-Trop2 ECD (R&D Systems, FAB1122A, FAB1122F, and BAF1122), anti-Myc tag (Cell Signaling, no. 2276S and no. 2278), anti Erk-2 (Santa Cruz Biotechnology, sc-154), anti-α-tubulin (Santa Cruz Biotechnology, sc-5286), anti-PS-1 (Cell Signaling, no. 5643S), anti-PS-2 (Cell Signaling, no. 2192), anti-cyclinD1 (Cell Signaling, no. 2926), anti-β-catenin (Transduction Laboratories, 610454), anti-EpCAM (Abcam, ab68892), and anti-EpCAM C terminus (Epitomics, 1144-1).

Techniques: In Vitro, Western Blot, Immunoprecipitation, Expressing, Staining, Confocal Microscopy, Transfection, Infection, Mutagenesis, Transduction, Isolation