Mechanosensitive Ion Channel Piezo1 Activated by Matrix Stiffness Regulates Oxidative Stress-Induced Senescence and Apoptosis in Human Intervertebral Disc Degeneration

Figure 2

Stiff ECM induces the Piezo1 expression and activates oxidative stress and ER stress. (a) Piezo1 mRNA expression levels were analyzed by qRT-PCR in human NP cells cultured in polystyrene plates with different stiffnesses (soft: 1 kPa, stiff: 25 kPa) for 6, 12, 24, and 48 h. (b) The Piezo1 expression and cell morphology after 24 h of treatment were evaluated using immunofluorescence. Cytoskeleton was stained by phalloidin, and Cell nuclei are stained by DAPI. (c, d) The Piezo1 protein level was measured by western blotting. (e) Piezo1 mRNA expression levels were analyzed by qRT-PCR. (f) Intracellular Ca²⁺ levels measured using the specific Ca2 + -sensitive fluorescent indicator Fura-4-AM and analyzed via fluorescence microscopy. (g, h) The ROS levels were measured using DCFH-DA and analyzed using a flow cytometer. (i, j) GRP78 and CHOP protein levels were evaluated by western blotting. Data are presented as (, , magnification: ×400, scale bar: 50 μm).

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