Fig. 3

Ex vivo live cell imaging of GFP-BAX translocation in retinal ganglion cells (RGCs) of retinal explants. Retinal explants were imaged 7 days after optic nerve crush (ONC) surgery. A Still images of a GFP-BAX expressing RGC going punctate during a live-cell imaging session (see supplemental video S1). The time stamp refers to the elapsed time from the initiation of the imaging session. DRAQ5 counterstaining was used to highlight nuclei. Scale bar = 5 µm. B Graph showing the conversion of imaged cells going from diffuse to punctate in retinas from nerve ONC relative to contralateral eyes (Con). Significantly more cells converted to punctate GFP-BAX in the ONC retinas compared to contralateral retinas during the imaging period (***P > 0.001, t-test), indicating that initiation of GFP-BAX translocation was primarily reliant on previous ONC. Each data point represents a single retinal explant. C Box and Whisker plots of the maximum rate of translocation of GFP-BAX in cells from retinal explants. In addition to explants from retinas harvested from eyes that had undergone ONC, we also imaged naïve retinas that were exposed to staurosporine (STS) for comparison. Each data point represents the rate obtained from 160 mitochondrial loci in 22 different cells for ONC and 52 loci from 10 cells for STS (see Table 1). The rate of GFP-BAX translocation is significantly slower in STS-treated cells than in ONC-induced cells (***P < 0.0001, t-test), but both these rates are significantly slower (P < 0.0001, individual t-tests) than the mean average translocation rate (-1.74 ± 0.8 Log₂(RFU/min) dashed lines on graph) measured from 4 different tissue culture cells lines (D407, HeLa, HCT116, 661W cells – see Table 1)