Enigma blocks Cbl-c-mediated RET9MEN2A ubiquitination.

<p>(<b>A</b>) HEK293T cells were transfected with RET9MEN2A along with GST-Cbl-c, FLAG-Enigma or FLAG-EnigmaΔLIM2-3 alone and in combination as indicated above the blots. All transfections included an equal amount of HA-ubiquitin. At 40h post-transfection, all cells were treated with 20nM MG-132 for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for RET and HA-ubiquitin. Co-immunoprecipitation of Cbl-c and Enigma were evaluated as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control. (<b>B</b>) RET truncation constructs used for mapping Cbl-c and Enigma interaction sites. Structural domains include the extracellular cadherin-like repeats (C1-4), a cysteine rich region (CR), a hydrophobic transmembrane region (TM), a split cytoplasmic tyrosine kinase domain (TK1 and TK2), and intracellular tyrosines subsequently mutated to an F or stop (*) to create each construct used in this study. (<b>C</b>) HEK293T cells were transfected with GFP-tagged Enigma both alone and in combination with RET9MEN2A or one of a series of RET9MEN2A mutant constructs as indicated above each blot. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for GFP-Enigma. Molecular weight standards are indicated on the left of each panel and Hsc70 serves as loading control. (<b>D</b>) HEK293T cells were transfected with each of the RET9MEN2A constructs both alone and in combination with GST-Cbl-c as indicated above each blot. All transfections were balanced with empty vector controls, and cells were collected 48 h post-transfection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. GST pull-downs were performed on 300 µg of each of the whole cell lysates and immunoblotted for Cbl-c, phosphotyrosine (pY), and RET as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control.</p

Cbl binds RET and MEN2A kinases.

<p>(<b>A</b>) HEK293T cells were transfected separately with pJ7Ω plasmids encoding each of the following four RET kinase isoforms: RET 9, RET 51, and their MEN2A (C634R) mutant isoforms. Each was transfected both with and without GST-Cbl-c. Whole cell lysates were collected at 48h post-transfection and GST pull-downs were performed on 300 µg of whole cell lysates and immunoblotted (IB) as indicated. Whole cell lysates were immunoblotted as indicated and molecular weight standards are indicated on the left of each panel β-actin serves as a loading control. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c both with and without RET9MEN2A. Replicate plates were treated with either 500 µM Sorafenib (+) or DMSO as vehicle control (-) for 90m prior to collection. Whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysates using anti-RET antibody with Protein A/G Sepharose and immunoblotted for RET and phosphotyrosine (pY) as indicated. GST pull-downs were performed, as described above, and immunoblotted (IB) for RET, phosphotyrosine (pY), and GST-Cbl-c as indicated. Molecular weight standards are indicated to the left of each panel and Hsc70 serve as loading controls.</p

Enigma abrogates RETMEN2A ubiquitination.

<p>(<b>A</b>) RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates described above and immunoblotted for HA-ubiquitin and phosphotyrosine (pY) as indicated on the right. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c with and without FLAG-Enigma along with either RET9MEN2A or RET51MEN2A. Cells were collected 48h post-transfection, and whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for RET and HA-ubiquitin as indicated on the right. Molecular weight standards are indicated on the left and Ponceau S staining serves as a measure of protein loading. (<b>C</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above the blots. All transfections were performed in triplicate. At 24h post-transfection, triplicate plates were pooled and replated. At 40h post-transfection, replicate plates were treated with 100ng/mL cycloheximide (CHX) for either 3 or 5h as indicated. Control plates received an equivalent volume of DMSO vehicle control for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates was immunoblotted as indicated. RET steady state levels were then assessed using β-actin as a loading control. (<b>D</b>) Densitometric analysis of RET steady state levels in the presence of GST-Cbl-c and Enigma, either alone or in combination. Levels were all compared to Vector transfected cells in the absence of cycloheximide. Error bars denote mean ± SE (n  = 3). (<b>E</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above each blot. At 24h post-transfection, all cells were starved of FBS for 24h prior to cell harvesting. A total of 20 µgs of each of the whole cell lysates were immunoblotted as indicated to the right of each panel. Phospho-MAPK steady state levels were assessed using MAPK and Hsc70 as loading controls. Molecular weight standards are indicated on the left of each panel. (<b>F</b>) HEK293T cells were transfected with wild-type RET and the RET co-receptor, GFRα1 along with GST-Cbl-c and Enigma both alone and in combination. Vector controls included empty GST vector and all transfections were performed in duplicate and included HA-tagged ubiquitin. At 24h post-transfection, duplicate plates were pooled and re-plated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 24h, then stimulated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087116#pone.0087116-Boulay1" target="_blank">[43]</a>, using 30ng/mL GDNF and 100ng/mL exogenous GFRα1 (+) or water control (-) for 15m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess RET ubiquitination, RET immunoprecipitations were performed on 300ugs of whole cell lysate and immunoblotted for HA-ubiquitin as indicated on the right. (<b>G</b>) HEK293T cells were transfected with EGFR along with GST-Cbl-c and Enigma both alone and in combination. All transfections were performed in duplicate. At 24h post-transfection, duplicate plates were pooled and replated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 8h, then stimulated with 100ng/mL EGF (+) or water control (-) for 10m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess EGFR ubiquitination, EGFR immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for EGFR and HA-ubiquitin as indicated on the right. Molecular weight standards are shown to the left of each panel and tubulin serves as loading control.</p

Adaptation of Laser Microdissection Technique for the Study of a Spontaneous Metastatic Mammary Carcinoma Mouse Model by NanoString Technologies

<div><p>Laser capture microdissection (LCM) of tissue is an established tool in medical research for collection of distinguished cell populations under direct microscopic visualization for molecular analysis. LCM samples have been successfully analyzed in a number of genomic and proteomic downstream molecular applications. However, LCM sample collection and preparation procedure has to be adapted to each downstream analysis platform. In this present manuscript we describe in detail the adaptation of LCM methodology for the collection and preparation of fresh frozen samples for NanoString analysis based on a study of a model of mouse mammary gland carcinoma and its lung metastasis. Our adaptation of LCM sample preparation and workflow to the requirements of the NanoString platform allowed acquiring samples with high RNA quality. The NanoString analysis of such samples provided sensitive detection of genes of interest and their associated molecular pathways. NanoString is a reliable gene expression analysis platform that can be effectively coupled with LCM.</p></div

Pathology annotations of target area and documentation of carcinoma dissection by laser cutting.

<p>(A) Target area, carcinoma (a), was annotated by study pathologist on a digital image of reference H&E section of mammary gland (b) by green line (arrow); (B) View of LCM section with target area (a) on the dissecting screen of MMI CellCut microdissection instrument; (C) Dissecting screen view of the carcinoma area with the laser cut path (arrow); (D) Dissecting screen view of carcinoma area after retrieval of target cutout. A: Scale bar corresponds to 650 μm; B-D: Scale bars correspond to 300 μm.</p

Unsupervised hierarchical clustering of normal mammary gland versus primary tumor.

<p>Scaled down representation of the entire cluster is based on 24 genes differentially expressed between normal mammary gland (NMG) and primary mammary tumors (PT). Each row represents a single gene and each column represents a sample. Red color indicates upregulation, green color—downregulation, and black color—no change in expression level compared with the reference sample.</p

Unsupervised hierarchical clustering of carcinoma versus EMT.

<p>Scaled down representation of the entire cluster is based on 17 genes differentially expressed between carcinoma (epithelial-like areas) and EMT (spindle-like areas). Each row represents a single gene and each column represents a sample. Red color indicates upregulation, green color—downregulation, and black color—no change in expression level compared with the reference sample. EMT: Epithelial-Mesenchymal transition.</p

Pearson Correlation Scores for Individual Sample Groups Between NanoString and Microarray (p-value < 0.001).

<p>Pearson Correlation Scores for Individual Sample Groups Between NanoString and Microarray (p-value < 0.001).</p

LCM Staining Protocol.

<p>LCM Staining Protocol.</p

Representative Agilent electropherograms of high quality RNA retrieved from the control sections and corresponding LCM targets.

<p>(A, C, F) Frozen section of normal mammary gland (A), primary mammary tumor (C) and lung metastasis (F) placed directly in lysis buffer for RNA extraction. (B) LCM sample of normal mammary tissue; (D, E) LCM cell populations of primary mammary tumor: carcinoma (D) and EMT (E); (G) LCM sample of lung metastasis.</p