Fig. 3

Protoplast shrinkage at 55 °C HS is an ATP- and Ca²⁺-independent process. a Mitochondria in BY-2 cells stained with MitoTracker Red and imaged 10 min after 55 °C or 85 °C HS, or after treatment with 48 μM CCCP under normal temperature. Severely damaged mitochondria were observed upon all three treatments. b Loss of intracellular ATP content upon HS. Snap freeze-thaw treatment in liquid nitrogen (N₂) and CCCP treatment were used as positive controls for completely disrupted and uncoupled mitochondria, respectively. The experiment was repeated twice, each time using four biological replicates per treatment. c MitoTracker Red staining of BY-2 cells exposed to 55 °C in the presence or absence of 15 μM Cyclosporin A (CsA) reveals that inhibition of MPTP opening does not rescue mitochondria from severe damage and loss of MMP caused by HS. d MitoTracker Red localization in the cells pre-treated with 10 mM EGTA prior to the HS reveals that chelation of extracellular Ca²⁺ does not rescue mitochondrial phenotype. e, f Dynamics of cell death (% SO-positive cells; e) and protoplast shrinkage (f) in cells with normal and uncoupled (48 μM CCCP treatment) mitochondria. g, h Pre-treatment with 10 mM EGTA before HS does not affect dynamics of cell death (% SO-positive cells; g) and protoplast shrinkage (h). Experiments shown in e–h were repeated three times, with ≥ 184 cells per treatment and time point. Each microscopy experiment was performed at least twice. Staining for no HS and 55 °C treatments were performed according to protocols i and ii, respectively (see “Sytox Orange and FM4-64 staining”). Scale bars, 20 μm (a) or 50 μm (c, d). IQR, interquartile range. b, e–h One-way ANOVA with Dunnet’s test; *p < 0.05