Fig. 4

Direct inhibition of nSMase indirectly increases mitochondrial superoxide and CTB uptake in naïve cells. A Cartoon of coculture system to detect transfer of CTB and MitoTracker from treated to naïve cells. B Diagram of drug treatment, CTB labeling, and mtSOX labeling of hRGC cocultures. C Representative images of live tdTomato-positive hRGCs (orange) where control (Ctrl) or GW4869-treated cells were labeled with CTB (green) and MitoTracker (magenta) and naïve cells were not acutely labeled with CTB and MitoTracker. Arrowheads highlight MitoTracker located in the extracellular space. D Confocal images of live DMSO- (top) and GW4869-treated hRGCs (bottom) identified by CTB labeling (green) cocultured with naïve cells. Prior to live imaging, cultures were incubated in mtSOX (magenta) to recover degraded mitochondria. Arrowheads indicate mtSOX localized in the extracellular space. E GW4869 significantly reduced MitoTracker labeling in treated cells (p = 0.0083). 112 Ctrl treated cells and 123 naïve cells from 11 replicates. 81 GW4869 treated cells and 125 naïve cells from 10 replicates. We did not detect a difference in MitoTracker labeling in vehicle and DMSO conditions (p ≥ 0.18) so data from both conditions were combined (Ctrl). F Quantification of accumulation of mtSOX in treated and naïve cells in Ctrl and GW4869-treated cocultures. GW4869 directly and indirectly (naïve) increased intracellular mtSOX fluorescence intensity compared to respective controls (p < 0.001). Ctrl coculture N = 32 Ctrl treated cells and 57 naïve cells from 10 replicates. GW4869 coculture N = 49 GW4869 treated cells and 80 naïve cells from 7 replicates. We did not detect a significant difference between vehicle and DMSO-treated cocultures, so datasets were combined (p ≥ 0.14). G Treatment with GW4869 directly (treated) and indirectly (naïve) increased CTB uptake (p ≤ 0.003). Ctrl coculture N = 84 Ctrl treated cells and 98 naïve cells from 21 replicates. GW4869 coculture N = 88 GW4869 treated cells and 91 naïve cells from 12 replicates. CTB uptake by cells in vehicle and DMSO-treated cocultures were similar and data were combined (p > 0.9999). H GW4869 significantly increased deposition of CTB in the extracellular space (p < 0.001). Ctrl N = 21 replicates, GW4869 N = 12 replicates. Statistics: (C, E, G) Kruskal–Wallis test, Dunn’s post-hoc comparisons, (F, H) t-test