Supplementary Figure 7: AT–56 effects on Schwann cells and neuronal survival, axon–Schwann cell interaction and myelination.

a) Graph showing the dose dependent effects of AT–56 treatment on Schwann cell survival, determined as percentage of Caspase 3⁺ cells over the total number of cells (3 coverslip/condition; 3 different experiments). 20 μM AT–56 does not alter Schwann cells survival. Error bars represent means ± s.e.m. (t–test analysis: untreated – 20 μM P = 0.1138; t=1.807, df=4; untreated – 50 μM P < 0.0001; t=7.028, df=7). N = 3 different experiments. b) Graph showing the dose dependent effects of AT–56 treatment on myelinating Schwann cell–DRG neuronal coculture, determined as number of MBP⁺ segments per field (3 coverslip/condition; 3 different experiments). Increased concentration of AT–56 impairs myelination. Error bars represent means ± s.e.m. (t–test analysis: untreated – 1 μM P = 0.3866; t=0.8806, df=26; untreated – 10 μM *** P <0.0001; t=5.651, df=31; untreated – 25 μM *** P <0.0001; t=6.736, df=22). N = 3 different experiments. c) Graph showing the dose dependent effects of AT–56 treatment on Schwann cell–DRG neuronal coculture survival. We counted the effects of AT–56 on Schwann cells and express it as percentage of Caspase 3⁺ Schwann cells over the total number of cells (3 coverslip/condition; 3 different experiments). 25 μM AT–56 does not alter Schwann cells survival. (t–test analysis: untreated – 25 μM p = 0.3874; t=0.9318 df=6). Error bars represent means ± s.e.m. N = 3 different experiments. d) Organotypic mouse DRG explants were maintained in the presence of 25 μM AT–56 for 7 days then stained for S100 (rhodamine) and β–tubulin (fluorescein). AT–56 treatment does not impair axon – Schwann cell association. N = 3 different coculture experiments. Bar: 50 μm.