Journal: Communications Biology
Article Title: A role of pigment epithelium-derived factor in zinc-mediated mechanism of neurodegeneration in glaucoma
doi: 10.1038/s42003-025-08370-8
Figure Lengend Snippet: Effect of zinc stress on PEDF secretion was studied in ARPE−19 ( a ) and Y79 ( b ) cells. a APRE−19 shows a slow response to zinc stress (half-maximum after 24 h), as confirmed by MTT test, as well as fluorescein diacetate/propidium iodide (FDA/PI) viability assay and confocal microscopy (Scale bar represents 25 μm, n = 3). b Zinc treatment decreased viability (half-maximal after 8 h) and induced apoptosis of Y79 cells, as evidenced by EZ4U test and flow cytometric annexin A5 assay ( n = 6). In both cases, zinc stress was accompanied by PEDF release ( p < 0.05). The amount of PEDF in the absence (Y79) or at 24 h (ARPE−19) of stress was taken as 100%. c-e Effect of zinc on the neurotrophic activity of PEDF was studied on SH-SY5Y cells ( c ) and Y79 c ells ( d-e ). c Preincubation of PEDF with zinc reduced its axogenic activity in respect to differentiated SH-SY5Y cells without affecting cell count (Scale bar represents 25 μm, n = 14). d Zinc inhibited the ability of PEDF to stimulate neuronal differentiation of Y79 cells (Scale bar represents 10 μm, n = 21). Neurites and nuclei were stained with anti-β-III tubulin ( c ) or synapsin ( d ) antibodies and Hoechst 33342, respectively, and visualized by confocal microscopy. e PEDF att e nuated Y79 cell death under oxidative stress but not zinc stress, as shown by LDH assay ( n = 3). Leaked LDH activity in the absence of stress was taken as 100%. f Phospholipase activity of PEDF-R in ARPE−19 cell membranes was monitored in the presence of apo or zinc-bound PEDF by quantifying fatty acid release from model phospholipid substrate using HPLC-MS/MS. Extracted ion chromatogram illustrating differences in linoleic acid ([M-H] - LA, m/z 279.2) formation in the presence of apo and zinc-bound PEDF. The histogram on the right illustrates the attenuation of PEDF-mediated enhancement of PEDF-R activity in the presence of zinc ( p < 0.05). PEDF-R activity without PEDF/zinc was taken as 100%. All data are presented as mean and error bars indicate SEM.
Article Snippet: The staining solution, containing 5 ml DMEM, 8 μl FDA (5 mg/ml) and 50 μl PI (2 mg/ml) was added to the wells, cells were incubated at room temperature for 4-5 minutes in the dark, washed with PBS and examined on Eclipse Ti-E confocal laser scanning microscope (CLSM) with A1 confocal module (Nikon Corporation, Japan) and CFI Plan Apo VC 20×/0.75 objective lens with a life support system, maintaining normal culturing conditions (37°C, 5% CO 2 ).
Techniques: Viability Assay, Confocal Microscopy, Activity Assay, Cell Counting, Staining, Lactate Dehydrogenase Assay, Tandem Mass Spectroscopy
![Effect of zinc stress on PEDF secretion was studied in ARPE−19 ( a ) and Y79 ( b ) cells. a APRE−19 shows a slow response to zinc stress (half-maximum after 24 h), as confirmed by MTT test, as well as fluorescein diacetate/propidium iodide (FDA/PI) viability assay and <t>confocal</t> <t>microscopy</t> (Scale bar represents 25 μm, n = 3). b Zinc treatment decreased viability (half-maximal after 8 h) and induced apoptosis of Y79 cells, as evidenced by EZ4U test and flow cytometric annexin A5 assay ( n = 6). In both cases, zinc stress was accompanied by PEDF release ( p < 0.05). The amount of PEDF in the absence (Y79) or at 24 h (ARPE−19) of stress was taken as 100%. c-e Effect of zinc on the neurotrophic activity of PEDF was studied on SH-SY5Y cells ( c ) and Y79 c ells ( d-e ). c Preincubation of PEDF with zinc reduced its axogenic activity in respect to differentiated SH-SY5Y cells without affecting cell count (Scale bar represents 25 μm, n = 14). d Zinc inhibited the ability of PEDF to stimulate neuronal differentiation of Y79 cells (Scale bar represents 10 μm, n = 21). Neurites and nuclei were stained with anti-β-III tubulin ( c ) or synapsin ( d ) antibodies and Hoechst 33342, respectively, and visualized by confocal microscopy. e PEDF att e nuated Y79 cell death under oxidative stress but not zinc stress, as shown by LDH assay ( n = 3). Leaked LDH activity in the absence of stress was taken as 100%. f Phospholipase activity of PEDF-R in ARPE−19 cell membranes was monitored in the presence of apo or zinc-bound PEDF by quantifying fatty acid release from model phospholipid substrate using HPLC-MS/MS. Extracted ion chromatogram illustrating differences in linoleic acid ([M-H] - LA, m/z 279.2) formation in the presence of apo and zinc-bound PEDF. The histogram on the right illustrates the attenuation of PEDF-mediated enhancement of PEDF-R activity in the presence of zinc ( p < 0.05). PEDF-R activity without PEDF/zinc was taken as 100%. All data are presented as mean and error bars indicate SEM.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5840/pmc12215840/pmc12215840__42003_2025_8370_Fig5_HTML.jpg)