Intracellular calcium response to four different agonists in MUG-Chor1 cells was measured.

<p>A) Histamine (His), acetylcholine (ACh), serotonin (5HT), and P2Y purinoceptor agonist mes-ATP were applied at a concentration of 10 µM for a period of 60 seconds as indicated by the grey bars; the ratio between 340 nm and 380 nm is given. After stimulation, the respective agonist was washed out for another 60 seconds. B) The first two peaks of ratio values of MUG-Chor1 cells indicate ACh-application in the presence of extracellular calcium (NT+[Ca²⁺]_ex; upper, open bar); peak reduction was induced by removing external calcium (NT w/o [Ca²⁺]_ex; upper, grey bar). Small bars represent the time of perfusion with ACh at the two different concentrations (10 µM: lower, grey bar; 100 µM: lower, black bar). C) Within the bar chart a summary of ACh-induced changes is given. The first two bars represent effects in [Ca²⁺]_i induced by ACh (10 µM; 100 µM) given as average value (± standard deviation) of the change in intracellular calcium concentration [Ca²⁺]_i (nM). The usage of calcium within the perfusion solution as well as the different concentrations of ACh is indicated below the x-axis. Significant changes evaluated by the students t-test are given (***, p<0.001; **, p<0.01).</p

HPF fixed vacuoles within the chordoma cells.

<p>All micrographs indicate the connection of the vacuoles. A) Overview of the vacuoles (V); arrows indicate the network within the MUG-Chor1 cells. B) Cells show a high number of small vacuoles connected to each other as well as endosomes and Golgi apparatus, surrounded by dense cytoplasm. C) Higher magnification enables the visualization of the linkages between either small vacuoles or endosomes and vacuoles; arrows indicate the exchange of material within the vacuoles.</p

Conventional electron microscopy of vacuoles in chordoma cells (arrows indicate the reduced connection of the vacuoles; mitochondria (M); nucleolus (N); vacuoles (V)).

<p>A) MUG-Chor1 cells show a prominent nucleus, abundant vacuoles with a diverse density of material. B) Higher magnification of large densely filled vacuoles. C) Overview of single vacuoles with barely visible contact zones between the vacuoles.</p

Structural association of mitochondrial membrane with the ER called MAM, represented by HPF.

<p>A) Abundant mitochondria within the cell, small connected vacuoles, and the connection of two chordoma cells with contact zones as well as excrescences (asterisk). B) Very close continuous contact zones of mitochondria (M) and ER. C) Connection of two chordoma cells with contact zones, excrescences (asterisk) and MAM complex.</p

Electron microscopy overview of the chordoma cells (stitched image).

<p>HPF fixation was used to demonstrate the heterogeneity of chordoma cells. Cell 1 (black line) showed a prominent nucleus (N) and connected vacuoles (V) in different sizes. Cell 2 (green line) and cell 3 (orange line) present fewer vacuoles, abundant mitochondria (M), and dense ultrastructure of the cytoplasm. Cell 4 (violet line) and cell 5 (blue line) show prominent vacuoles surrounded by a small cytoplasm matrix, containing mitochondria. Cell 6 (yellow line) exhibit small vacuoles and less dense cytoplasma.</p