Monensin

Genistein, chloroquine, monensin, nocodazole (cat no. 1228) were purchased from Tocris Bioscience (Bristol, UK). The recombinant rh-α-Gal-A enzyme was from commercial source: “Fabrazyme” from Sanofi/Genzyme Corporation (Cambridge, MA, USA). Biochemical and pharmacological characteristics of commercial rh-α-Gal-A described [18 (link)]. We used rh-α-Gal-A from leftover vials after reconstitution for patient use.

Lgl1 cKO OPC were treated with differentiation medium containing 10 μM Monensin (TOCRIS, 5223) for 4 h. Cells were monitored under TIRF for NG2 recycling monitoring during the first hour of treatment or were switched back to normal differentiation medium for 5 days before being fixed and immunostained for O4 and NG2. Endocytosis of the proteoglycan NG2 was followed using a mouse-anti-NG2.EC antibody against the extracellular domain of NG2 (courtesy of Dr. Stallcup). OPC cultures were incubated 24 h prior to live imaging with CellLight^® Reagents BacMam 2.0 (Molecular Probes) targeting the early endosome-specific protein Rab5a (ref C10586) of lysosome-specific protein Lamp1 (ref C10507) at a concentration of 50 PPC. Cells were starved 2 h prior to imaging in NBA medium, and 50 ng/mL of PDGF-AA was added right before imaging to induce endocytosis. Time-lapse movies were taken over time spans of 30–45 min with 30 s interval between the images to follow early endocytosis of NG2 antibody and 2–3 h with a 30 min interval to follow its internalization to the lysosome. After live imaging, cells were fixed 10 min in PFA4%, stained with the appropriate antibodies and imaged with a Nikon Ti Yokogawa CSU-22 spinning disk confocal equipped with a Plan Apo VC 60×/1.4 oil objective.