Figure 1: The localization of LKB1 is essential for proper SC myelination.

(a) A schematic illustration of the possible conservation and multifaceted roles for the Par polarity proteins during SC development and myelination. (b) Immunostaining of LKB1 (red) in purified SC-DRG cocultures illustrates that LKB1 is diffusely localized and enriched at SC–axon interface. Immunostaining of neurofilament illustrates the position of the neuronal axon (green). (c) Phosphorylated LKB1 (red) at ser-431 is asymmetrically localized to the SC–axon interface. (d) Phosphorylated LKB1 (red) co-localizes with Par-3 (green) at the SC–axon interface. The cell nuclei are detected by DAPI (blue). Scale bars=10 μm. (e) Western blot analysis of SC-DRG cocultures before and after induction of myelination by addition of ascorbic acid (denoted by the I). Western blots were probed with antibodies to phosphorylated ser-431 LKB1, LKB1, Oct-6 (a transcription factor expressed by premyelinating SCs) and the myelin protein P0. β-actin serves as a loading control. The asterisks indicate the concomitant expression of phosphorylated ser-431 with the myelin protein P0. (f) Floxed-LKB1 SC-DRG cocultures were established and SCs were transduced with a retrovirus to express Cre-recombinase and GFP (green). LKB1 immunostaining illustrates the knockdown of LKB1 expression in the transduced SCs. Arrows indicate the asymmetric localization of LKB1 in uninfected SCs. (g) Deletion of LKB1 in SCs disrupts the localization of Par-3 to the SC–axon interface. The arrow indicates the asymmetric localization of Par-3 (red) in the uninfected SC. The nuclei are detected with DAPI (blue). Scale bars=10 μm. (h) SC-DRG cocultures were established from crossing the floxed-LKB1 mice with the CNP-Cre mice and induced to myelinate for 10 days. Myelin internodes are identified with MBP staining (red) and nuclei are detected with DAPI (blue). Scale bar=100 μm. (i) Western blots from the SC-specific LKB1 knockout cocultures were probed with phos-ser-431 LKB1, LKB1, Oct-6 and the myelin protein P0. Representative western blots are displayed and all results were obtained from a minimum of three independent experiments (n=3).