Fig. 6
Autophagy is a critical mediator of neuroprotection conferred by Slc38a1 deficiency. a, b Assessment of the neuroprotective effect against ischemic stress conferred by Slc38a1 deficiency. Control or Slc38a1-null neurons were cultured under normal (anaerobic glucose deprivation [OGD]) or OGD conditions and stained with an anti-MAP2 antibody (a, n = 3) or PI (b, n = 6) to evaluate dead neurons. Bars = 100 µm. c–g Increases in the mRNA levels of autophagy-related genes associated with Slc38a1 deficiency. Total RNA was extracted from control or Slc38a1-null neurons, and the mRNA levels of each indicated gene were measured using qRT-PCR. Values were normalized to those of Gapdh (n = 6). h Evaluation of autophagy under ischemia. Cell-free lysates of Slc38a1-null neurons cultured under OGD were subjected to western blotting using anti-phospho-p62 and anti-actin antibodies (n = 4). i, j Suppressive effect of autophagy inhibitors on neuroprotection induced by Slc38a1 deficiency. Control or Slc38a1-null neurons were treated with 1 nM bafilomycin i or 5 µM chloroquine j in the presence or absence of OGD. Neuronal cell death was assessed using PI staining (n = 3)
