Journal: Oncogene

Article Title: Tumor microenvironment defines the invasive phenotype of AIP -mutation-positive pituitary tumors

doi: 10.1038/s41388-019-0779-5

Figure Lengend Snippet: Macrophage-conditioned media induce EMT-like phenotype in GH3- Aip -KD cells. a Macrophage-conditioned media (MCM) induces an EMT-like phenotype. Morphological changes in GH3- Aip -KD and control GH3-NT (representative phase contrast images, top panels) and quantification of cell morphology (bottom panels). Untreated and mesenchymal-to-epithelial transition state (replaced with DMEM) GH3- Aip -KD and GH3-NT cells showed an epithelial cobblestone-like morphology (phase contrast images: first, third, fourth, and sixth panels) whereas MCM-treated cells both GH3-NT and GH3- Aip -KD become spindle shaped and show mesenchymal like morphology (phase contrast images: second and fifth panels). Untreated GH3-NT and GH3- Aip -KD cells show no significant differences in cell size and shape. Morphology of the cells was quantified using six different parameters (ImageJ). Around 100 cells from each condition were evaluated. There was an increase in cell area ( b ) and perimeter ( c ) in MCM-treated GH3-NT and GH3- Aip -KD cells, while, cells without MCM treatment showed no difference. d Feret’s diameter (measure of cell elongation) was significantly higher in MCM-treated cells, indicating more elongated cell shape. e , f Circularity and roundness (a value closer to one is indicate more circular/rounded cells and close to zero indicated an elongated shape): untreated and mesenchymal-to-epithelial transition state cells of GH3-NT and GH3- Aip -KD showed more circular and rounded shape than MCM-treated cells. g Solidity, defined as the ratio of cell area to the enclosing convex polygon area, indicates the stiffness and deformability of cells, was decreased in both GH3-NT and GH3- Aip -KD cells undergoing EMT. Overall P value for multiple comparison is shown in the left upper corner of the graphs, while significance between groups are marked with *, <0.05, ***, <0.001 (two-way ANOVA followed by Newman–Keuls multiple comparison test). n = 3, performed in triplicates on three independent days. Scale bar = 25 µm

Article Snippet: We used rat pituitary cell line GH3 cells (obtained from European Collection of Authenticated Cell Cultures at the start of the project) and generated two stable knockdown cell lines, a 50% and an 80% knockdown, and a non-targeting control (GH3-NT) ( ).

Techniques: Control, Comparison

Journal: Oncogene

Article Title: Tumor microenvironment defines the invasive phenotype of AIP -mutation-positive pituitary tumors

doi: 10.1038/s41388-019-0779-5

Figure Lengend Snippet: Alterations of the EMT markers in GH3-NT and GH3- Aip -KD cells. a Immunofluorescence analysis of E-cadherin, ZEB1, and actin in GH3-NT and GH3- Aip -KD cells with or without MCM at 72 h. Untreated cells of GH3-NT and GH3- Aip -KD cells show membranous localization of E-cadherin and low level of cytoplasmic ZEB1 expression, while MCM-treated cells show lack of membranous but increased cytoplasmic E-cadherin expression and a significant increase in nuclear and cytoplasmic ZEB1 expression. After removal of MCM and culturing cells in 10% DMEM for 72 h, the cells revert back to their cobblestone-like morphology (mesenchymal-to-epithelial transition state), increased E-cadherin expression and localization as well as reduced ZEB1 expression. Actin staining revealed that untreated cells show cortical rings of actin. MCM-treated GH3-NT cells show granular pattern of actin with less actin stress fibers while GH3- Aip -KD cells showed prominent actin stress fibers and numerous actin spikes (inset). In contrast, in mesenchymal-to-epithelial transition state cells are gradually return to the original state of their actin cytoskeleton (×63 magnifications). DAPI was used to stain the nuclei; the pictures are representative of at least three experiments. b Immunoblotting (densitometric analysis and representative images) suggested that E-cadherin levels were significantly decreased and ZEB1 significantly increased in MCM-treated GH3- Aip -KD cells compared to GH3-NT cells. Overall P value for multiple comparison is shown, while significance between groups are marked with **, <0.01, ***, <0.001; two-way ANOVA followed by Newman–Keuls multiple comparison test. c Invasion assays showing that MCM treatment increases the invasion of GH3-NT and GH3- Aip -KD cells. Bar charts show the mean number of invading cells through the Matrigel-coated transwell chambers measured after 72 h. Both GH3-NT and GH3- Aip -KD cells show significant increase in invasion compared to the untreated cells, but was more significant in GH3- Aip -KD cells. Representative photographs of invading cells are shown (×10), right panels. P values indicated *, <0.05, **, <0.01; two-way ANOVA followed by Newman–Keuls multiple comparison test. Data represent mean values of three independent experiments

Article Snippet: We used rat pituitary cell line GH3 cells (obtained from European Collection of Authenticated Cell Cultures at the start of the project) and generated two stable knockdown cell lines, a 50% and an 80% knockdown, and a non-targeting control (GH3-NT) ( ).

Techniques: Immunofluorescence, Expressing, Staining, Western Blot, Comparison

Journal: Oncogene

Article Title: Tumor microenvironment defines the invasive phenotype of AIP -mutation-positive pituitary tumors

doi: 10.1038/s41388-019-0779-5

Figure Lengend Snippet: The role of the CCL5 pathway in macrophage migration. a Migration assays showing that macrophage migration was significantly reduced towards GH3- Aip -KD cell-derived conditioned media compared to GH3-NT control cell-derived conditioned media. Macrophages were in vitro treated with maraviroc (200 nM) for 24 h and the migration in response to GH3- Aip -KD-conditioned media and GH3-NT conditioned media was evaluated. Cells were counted in nine random fields and data are presented as mean ± SEM, n = 3. Graph showing the percentage of the MVC-treated migrated macrophages towards the GH3-NT and GH3- Aip -KD-conditioned medium relative to the vehicle. V vehicle, MVC maraviroc. P values indicated ***, <0.001; t -test. b Immunohistochemical analysis of CCL5 in normal pituitary (NP, n = 11), AIP- mutation-positive somatotroph adenomas ( AIP pos GH, n = 12) and sporadic somatotroph adenomas (Sp GH, n = 17). Graph showing that CCL5 is highly upregulated in AIP pos tumors compared to the normal pituitary (left panel). Right panels show the representative images of CCL5 staining. P values indicated **, <0.01; one-way ANOVA with Bonferroni multiple comparison test. All images are ×200 magnification and scale bar = 100 µm. c Immunohistochemical analysis of FLI1 in normal pituitary (NP, n = 11), AIP- mutation-positive somatotroph adenomas ( AIP pos GH, n = 12) and sporadic somatotroph adenomas (Sp GH, n = 17). Graph showing that FLI1 is highly upregulated in AIP pos tumors compared to the normal pituitary (left panel) and Sp GH tumors (left panel). Right panels show the representative images of FLI1 staining. P values indicated ***, <0.001; one-way ANOVA with Bonferroni multiple comparison test. All images are ×200 magnification and scale bar = 100 µm

Article Snippet: We used rat pituitary cell line GH3 cells (obtained from European Collection of Authenticated Cell Cultures at the start of the project) and generated two stable knockdown cell lines, a 50% and an 80% knockdown, and a non-targeting control (GH3-NT) ( ).

Techniques: Migration, Derivative Assay, Control, In Vitro, Immunohistochemical staining, Mutagenesis, Staining, Comparison