Journal: Cell Death & Disease
Article Title: Stress-induced precocious aging in PD-patient iPSC-derived NSCs may underlie the pathophysiology of Parkinson’s disease
Figure Lengend Snippet: NSCs derived from patient-specific PD iPSCs manifested premature aging phenotypes and were sensitive to irradiation. a iPSC-derived neural stem cells were stained positively for NSC-specific markers Nestin and SOX2. Scale bar: 100 μm. b , c Neural differentiation capacity was severely impaired in PD iPSCs. b The representative images for spontaneous differentiation of PD- and WT-NSCs toward neurons and astrocytes as determined by the neuronal marker (Tuj1) and the astrocyte marker (GFAP). c Statistic results showed the percentage of Tuj1 and GFAP cells out of all cells derived from WT- and PD iPSCs. Scale bar: 100 μm. d – f The proliferative capacity of PD-NSCs was compromised as revealed by CCK8 assays and Ki67 staining. d Cell growth curve was detected by CCK8 assays at 24, 48, 72, 96, and 120 h. e Proliferation capacity was determined by Ki67 staining. f Graph showed the percentage of Ki67 cells. Scale bar: 100 μm. g , h Cellular senescence was significantly aggravated by IR treatment as evaluated with the SA-β-gal assay. g Representative images from at least three independent experiments were presented. h The statistical results showed the percent of SA-β-gal-positive cells (%). Scale bar: 100 μm. i , j ROS level increased significantly in PD-NSC with IR treatment. i Cellular ROS was stained with DCF-DA and observed under a fluorescence microscope. j ROS level was detected by fluorescence microplate reader. Scale bar: 100 μm. k , l Proliferative capacity was further decreased in PD-NSCs after IR treatment as revealed by Ki67 staining. k Expression of Ki67 in cells with or without IR treatment. l Graph showed the ratio of Ki67-positive cells. Scale bar: 100 μm. m , n DNA damage accumulation increased dramatically in PD-NSCs by IR as revealed by γH2AX staining (red) at 48 h post IR treatment. m Formation of DNA double-strand breaks in NSCs post irradiation. n Graphical depiction indicated the number of γH 2 AX foci in NSCs. Scale bar: 10 μm. o Cell proliferation curve of WT-NSCs and PD-NSCs with or without IR. Cell numbers were analyzed at 24, 48, 72, 96, and 120 h. p The expression level of cell senescence markers p53, p21, and p16 was augmented in PD-NSCs with IR treatment, analyzed by western blotting and densitometry. NSCs were treated with 10 Gy of IR and incubated for the indicated periods. β-actin was used as the loading control. All the data were expressed as mean ± SD. * P
Article Snippet: SA-β-gal assay Cellular senescence was determined using the SA-β-gal staining Kit (Genmed Scientifics).
Techniques: Derivative Assay, Irradiation, Staining, Marker, β-Gal Assay, Fluorescence, Microscopy, Expressing, Western Blot, Incubation