Fig. 1: Validation of a cellular model of intracellular iron accumulation and induction of cell death. | Cell Death & Disease

Fig. 1: Validation of a cellular model of intracellular iron accumulation and induction of cell death.

From: Autophagy deficiency exacerbates iron overload induced reactive oxygen species production and apoptotic cell death in skeletal muscle cells

Fig. 1

Time-lapse images examining intracellular iron level in L6 skeletal muscle cells, using PGSK. Cells were treated with 250 µM FeCl3 for up to 60 min concurrently with PGSK dye. A Representative images taken at time 0 and 60 min without or with FeCl3 treatment and B quantification of PGSK mean fluorescence intensity from images taken every 5 min (n = 3). C shows representative images obtained using FRET iron probe 1 (FIP‐1) without or with FeCl3, 250 μM for 24 h and quantification of FIP-1 (Green/FRET ratio) is shown in D (n = 3). Quantification of caspase 3/7 activity was assessed using an activatable fluorescent substrate done by CellInsight CX7 to examine dose- and time-dependent apoptotic cell death with a dose‐response to FeCl3 (50, 100, 150, 200 and 250 µM) shown in E and at multiple time points (250 µM FeCl3, at 1, 2, 4, 8, 24 h) in F (n = 3). To verify these observations, western blot analysis of cleaved caspase 3 was determined after treatment of cells with 250 µM FeCl3, at 1, 2, 4, 8, 24 h G and quantification is shown in H (n = 5). Scale bar in A 50 µm and C 100 µm. All graphs show mean ± SEM and * = P < 0.05, ** = P < 0.01, *** = P < 0.001 versus control.

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