Live Cell Imaging Supports a Key Role for Histone Deacetylase as a Molecular Target during Glioblastoma Malignancy Downgrade through Tumor Competence Modulation

Figure 5

iHDAC treatment disrupts voltage-dependent Ca⁺⁺ channels signaling by live cell imaging. iHDAC-treated U87-MG cells (100 nM TSA for 72 hours) were incubated with Fura 2-AM and analyzed by confocal microscopy. Significative differences in the basal levels of intracellular Ca²⁺ were not detected among the experimental groups (A–D). The fluorescence of 100 cells was continuously monitored for approximately 10 minutes in a fluorescence microscope (E-F). DMSO (E–G) or iHDAC (F–I) treated cells were continuously perfused with fluorimetry solution and stimulated with 20 mM KCl and 50 mM ATP. The variation of intracellular Ca⁺⁺ ([Ca²⁺]I) was evaluated by the fluorescence emitted at 488 nm. The responsiveness to KCL and ATP was quantified, and U87-MG cells treated with iHDACs were less responsive to KCl and ATP than DMSO-treated cells (J). Qualitative RT-PCR revealed constitutive expression of P2X7 in both control and iHDAC-treated groups. On the contrary, the alpha subunit 1H of the CACNA T-type calcium channel is not expressed by U87-MG cells. .