Extended Data Fig. 9. Additional and control studies for neuro-regeneration experiments.

a, Top panel; Control experiment to test the biological activity of 1938 post-freezing. An aliquot of 100 μM 1938 stock solution in dH₂O and vehicle was defrosted and tested for induction of pAKT^S473 by 15 min treatment of A549 cells, using insulin (1 μM) or 1938 (10 μM from control stocks in DMSO) as positive controls. Bottom panel; pAKT^S473 induction in exposed sciatic nerves, injected with vehicle (autoclaved H₂O) or 1938 (from stocks in autoclaved H₂O) or bathed in a solution of vehicle or 1938. After 30 min, the nerves were washed and processed for analysis as described in Materials and Methods. Cell extracts of MCF7 breast cancer cells stimulated for 15 min with 5 μM 1938 or vehicle (DMSO) were loaded on the gels as positive controls. n=1 experiment. b, Representative immunohistochemistry images of a transverse section through the distal common peroneal rat nerve, showing ChAT- and neurofilament-positive axons with tissue architecture typical of normal tissue. Scale bar = 50 μm. c, Representative immunohistochemistry images of rat TA muscle, showing a α-BTX-stained post-synaptic neuromuscular structure with associated neurofilament-positive neurons. Scale bar = 20 μm. n=5 animals.