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Fig. 8 | Acta Neuropathologica Communications

Fig. 8

From: Chemokines modulate the tumour microenvironment in pituitary neuroendocrine tumours

Fig. 8

a Morphological evaluation of GH3 cells after treatment for 72 h with complete medium and RAW 264.7 macrophage-CM, either from untreated macrophages (−PMA_Raw.CM) or macrophages treated with PMA (+PMA_Raw.CM). Data are shown as mean ± standard error of the mean (SEM) for the 6 morphological parameters evaluated by Image J: cell area (μm2), Feret’s diameter (μm), solidity (0–1), perimeter (μm), roundness (0–1) and circularity (0–1). Seventy-five cells were analysed per experiment, with a minimum of 3 experiments per treatment condition. Scale bar 25 μm. ***, <0.001 (one-way ANOVA with Bonferroni multiple comparison test). Alterations on actin cytoskeletal fibers in GH3 cells after treatment with macrophage-CM for 72 h in comparison to complete medium; representative images taken on confocal microscope at 63x magnification; DAPI was used to stain the nuclei. b Matrigel-coated chamber invasion assays on GH3 cells towards complete medium, −PMA_Raw.CM and + PMA_Raw.CM after 72 h. Data are shown as mean ± SEM for the ratio of invading GH3 cells towards -PMA_Raw.CM and + PMA_Raw.CM in relation to invading GH3 cells in complete medium conditions. Invasion studies were repeated 4 times in duplicate. *, <0.05 (one-way ANOVA with Bonferroni multiple comparison test). c MMP-9 expression in GH3 cells in complete medium or after treatment for 24 h with + PMA_Raw.CM, as determined by RT-qPCR. Data are shown mean ± SEM for the relative MMP9 mRNA fold change expression to GAPDH, as determined by the ∆∆CT method. n = 3. **, <0.01 (Mann Whitney U test). d Alterations in E-cadherin and ZEB1 expression by GH3 cells after 72 h treatment with complete medium, −PMA_Raw.CM or + PMA_Raw.CM. Untreated GH3 cells show strong E-cadherin with membranous localisation but also in the cytoplasm as well as low nuclear ZEB1 expression, while macrophage-CM treated GH3 cells display decreased E-cadherin expression and increased nuclear ZEB1 expression. Pictures were taken on confocal microscope at 63x magnification. DAPI was used to stain the nuclei. E-cadherin and ZEB1 fluorescent intensities are shown as mean ± SEM and were quantified in 30 different cells per treatment condition. ***, <0.001 (one-way ANOVA with Bonferroni multiple comparison test)

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