Fig. 4: Nuclear confinement-triggered mitochondrial fragmentation is dependent on Drp1.
From: Mechanical confinement induces ferroptosis through mitochondrial dysfunction
a Representative time-lapse images of mitochondria (Mito Green) in a HeLa cell compressed by AFM. The position of the tipless probe is indicated by the yellow dotted box. Insets are magnified images of areas indicated by white boxes. Scale bar = 10 μm. b Representative time-lapse images of mitochondria (Mito Red) in intact HeLa cells or cytoplasts during 90 min confinement mediated by microspheres. Insets are magnified images of areas indicated by white boxes. Scale bar = 10 μm. c Representative live-cell images of mitochondria (Mito Red) in control (shCtrl) and Drp1 knockdown (shDrp1) HeLa cells upon confinement mediated by microspheres. Scale bar = 10 μm. d The percentage of cells with fragmented mitochondria in shCtrl and shDrp1 HeLa cells upon 90 min confinement, related to (c) (n(shCtrl) = 56 cells, n(shDrp1) = 49 cells, N = 3 biologically independent experiments, ****p < 0.0001, two-sided Fisher’s exact test). e Representative time-lapse images of mitochondria (Mito Green) in HeLa cells expressing mCherry-Drp1-K38A during 60 min confinement mediated by microspheres. Non-transfected HeLa cells in the same sample were used as a control. Scale bar = 10 μm. f The percentage of cells with fragmented mitochondria in HeLa cells expressing mcherry-Drp1-K38A or in the control group upon 60 min confinement, related to (e) (n(K38A) = 50 cells, n(Control) = 47 cells, N = 3 biologically independent experiments, ****p < 0.0001, two-sided Fisher’s exact test). g Total ROS level in shCtrl and shDrp1 HeLa cells in the absence (Control) and presence (Confined) of 4 h confinement mediated by microspheres (n = 50 cells, N = 3 biologically independent experiments, ns represents not significant, ****p < 0.0001, unpaired two-way ANOVA with Sidak’s test). h Lipid ROS level in shCtrl and shDrp1 HeLa cells in the absence (Control) and presence (Confined) of 5 h confinement mediated by microspheres (n = 40 cells, N = 3 biologically independent experiments, ns represents not significant, ****p < 0.0001, unpaired two-way ANOVA with Sidak’s test). i The percentage of cell death in shCtrl and shDrp1 HeLa cells upon 9 h confinement mediated by microspheres (n = 8 independent fields, N = 3 biologically independent experiments, mean ± SD, ****p < 0.0001, two-sided unpaired Student’s t test).
