SUPPLEMENTARY DATA
In a supplementary set of experiments, observations of phenotype of cells and nucleus, through bright field and confocal microscopy was done.
Methodology
Examination of cell morphological change
Cell morphology was examined using phase-contrast light microscopy (Nikon TE 2000 Eclipse microscope).
For morphological analysis of nucleus and nuclear condensation, after treatment for 48h, PBS washing of cells followed by fixation in 3.7% paraformaldehyde in PBS at 4 °C for 15 min was done. After washing with cold PBS and then PBS containing 5μg/ml DAPI was added for 20 min.
The morphology of the cell's nuclei was examined using a Zeiss 510 Confocal laser-scanning microscope fitted with an argon-ion laser at an excitation wavelength of 350 nm. Nuclei were measured to have the regular phenotype as they were bright bright and homogenously. Apoptotic nuclei were recognized by the condensed nuclear size and chromatin gathering at the periphery of the nuclear membrane or total destruction morphology of the nuclear bodies.
Results:
Bright field images result showed that pretreatment Spirulina extract (1μg/mL) pretreatment noticeably enhanced cell morphology following HG treatment. HG challenge caused visibly morphological changes, as well as applanate and shrunken shapes with loss of cellular integrity when compared with NG untreated control. However, Spirulina pretreatment alone did not induce cell morphological changes (Fig.1a S).
To observe the nuclear morphology and cell death, H9c2 cells were stained with DAPI nuclear dye. The compression of nuclei and nuclear fragmentation, which are the character of apoptotic nuclei, were clear after exposure to HG (33mM) for 48 h. The confocal images of cells exposed to HG alone displayed apoptotic features in the nuclei. The attributed changes of the apoptotic nuclei were significantly reduced in the cells pretreated with Spirulina extract (1μg/mL) (Fig.1b S). However, isolated Spirulina extract (1μg/mL) pretreatment did not induce any nuclear changes.