Fig. 2

Mechanical stress affects ferroptosis through Piezo1 activation. a Detection of intracellular ROS in NPCs treated with GsMTx4 or Fer-1 under 1 MPa mechanical stress for 24 h in a Ca²⁺-free medium. using DCFH-DA and quantitative analysis of relative MFI (n = 3). b Representative transmission electron microscopy (TEM) images of NPCs treated with different stimulations for 24 h. Arrows indicate shrunken mitochondria. Scale bars, 20 μm (Low field), 5 μm (High field). c The mitochondrial function was detected using Mitotracker Kit. d WB analysis of mitochondrial function markers OPA1, Mfn2, Mfn1 and DRP1 of NPCs treated with or without GsMTx4 under 1 MPa mechanical stimulation for 24 h and quantification. GAPDH was used as an internal control (n = 3). e JC-1 assay showing mitochondrial membrane potential of NPCs. JC-1 monomer was stained green, and JC-1 aggregates were stained red. f WB analysis of iron metabolic markers and ferroptotic genes of NPCs. g PCR analysis of iron metabolic genes Tfrc, Fpn, Fth1 and ferroptotic genes Acsl4, Fsp1, Gpx4 of NPCs treated with or without GsMTx4 under 1 MPa mechanical stimulation for 24 h and quantification (n = 3). h WB analysis of iron metabolic markers and ferroptotic markers of NPCs treated with or without Fer-1 under 1 MPa stress for 24 h (n = 3). i, j Quantitative analysis of relative MFI (n = 3). k Representative histogram plot for fluorescence of oxidized BODIPY-C11. l Lipid ROS in NPCs treated with or without GsMTx4 under 1 MPa mechanical stress for 24 h by using BODIPY-C11. All data are expressed as the mean ± SEM, n = 3 replicates from one representative of 3 independent experiments. ns (no significance), *P < 0.05, **P < 0.01, ***P < 0.001