Fig. 3: Wireless electrical–molecular communication mediated by a.c.-EF-responsive bio-nanoantennae induces caspase-3/7-mediated apoptosis in preclinical GBM cells.
From: Wireless electrical–molecular quantum signalling for cancer cell apoptosis
a–c, Metabolic activity of GIN/GCE 31, GIN/GCE 28 and cortical astrocytes was analyzed using a PrestoBlue HS assay. GIN cells, GCE cells and human cortical astrocytes were treated with [email protected] c@Z for 8 h followed by a.c. EF stimulation (3 MHz, 0.65 V cm–1) for 12 h. The control is no treatment with either bio-nanoantennae or a.c. EFs. Error bars represent mean ± standard error of mean (s.e.m.) obtained from triplicate experiments repeated three times. Statistical analysis was performed by applying a two-way ANOVA with a Tukey’s post-test. d–g, Representative samples from flow cytometric analysis of cells stained with CellEvent Caspase-3/7 Green to detect caspase 3/7 apoptotic activity and Zombie NIR fixable dye to detect the dead cell population. The quadrants in the figure represents the following: A-D-, no cell death either due to caspase 3/7 apoptosis or necrosis; A+D-, caspase 3/7 positive apoptotic cells; A+D+, non-viable/dead cells caused by apoptosis; and A−D+, necrotic cells. The gating strategy is shown in Supplementary Fig. 14. FITC, fluorescein isothiocyanate and NIR, near infra-red are filters. h, High-magnification confocal microscopy images to demonstrate caspase 3/7 activation immediately after the treatment with a.c. EFs (3 MHz, 0.65 V cm–1) for 12 h in the presence of bio-nanoantennae. Cells were fixed with paraformaldehyde followed by counterstaining with a caspase 3/7 detection kit (green), Cytopainter actin phalloidin (Texas Red 591, in red) and Hoechst nuclear stain (blue). Scale bars, 20 µm. To confirm caspase 3/7 activation, at least five confocal images (each with nearly 30 cells) were taken at ×10. High-resolution (×63) images show both morphology and caspase 3/7 activation (in green). The –EF and +EF indicate before and after the EF treatment. i, Confocal microscopy images to demonstrate the cytoplasmic localization of [email protected] c@Z immediately after the treatment with a.c. EFs (3 MHz, 0.65 V cm–1) for 12 h in the presence of bio-nanoantennae. Cells were stained with late endosome dye (green) and imaged using a Leica confocal microscope with green fluorescent protein, GFP (late endosomes) and Alexa 633 ([email protected] c@Z) filter settings. Scale bars, 100 µm. White arrows represent the endosomal escape and localization of bio-nanoantennae, at least 60 cells were analysed.
