FIGURE 1.

CQ, Baf A1, and starvation result in elevated autophagy-associated proteins. A, whole cell lysates from C17.2 cells were changed to fresh media with or without CQ for 6, 12, and 24 h followed by Western blot analysis. CQ exposure resulted in increased LC3-II levels as early as 6 h and continued to increase over time compared with UT cells. B, Beclin1 levels were assessed via Western blot analysis on C17.2 whole cell lysates that were treated with CQ as described above. CQ-treated cells at 24 h contained elevated Beclin1 protein levels compared with UT, 6-h, and 12-h lysates. Western blots were digitized by UN-SCAN-IT software, averages for LC3-II/β-tubulin and Beclin1/β-tubulin pixel totals were calculated, and the data points were expressed in -fold change. Data points in A and B represent the mean ± S.E., with n = 4. *, p < 0.01 by one-way ANOVA/Bonferroni's post hoc test versus untreated controls. C, C17.2 NPCs were co-transfected with LC3-GFP and LAMP1-cherry and treated for 6 h with lysosomal dysfunction-inducing agents CQ or Baf A1 or subjected to serum starvation for 3 h. Autophagic stress leads to the accumulation of autophagosomes as evidenced by the presence of intense, punctate GFP-LC3 in NPCs treated with CQ, Baf A1, or starvation (Stv) compared with the weak, diffuse GFP-LC3 signal observed in UT cells. CQ and starvation resulted in increased dual LC3-GFP and LAMP1-cherry co-labeled autophagolysosomes (white arrowheads) in contrast to Baf A1 and untreated NPCs. Scale bar, 20 μm.