Figure 3. MB promotes AV formation and maturation. Primary neuronal cultures from GFP-LC3 mice were exposed to DMSO vehicle (A) or 0.02 μM MB (B) for 6 h. (C) shows quantification of the results. MB treated cells (gray bar) had significantly more puncta compared with vehicle-treated cells (black bar). In addition, the number of puncta per cell was greater following MB treatment (D). An additional group of primary neurons were incubated with 0.02 μM MB or DMSO control for 6 h. Following treatment, media was exchanged and cells were maintained in culture for a further 4 h to allow the MB to wash out. No significant difference was observed in the number of puncta in vehicle treated (E) vs. MB treated (F) cells. CHO cells transfected with a GFP-RFP-LC3 construct were treated with vehicle (G), 1 μM bafilomycin (H) or 0.01 μM MB (I). GFP and RFP images were collected and the percentage of GFP+/RFP+ positive AVs (yellow bars) and RFP+/GFP- positive autophagolysosomes (red bars) per cell were determined (J). MB treated cells had a significantly higher percentage of RFP+/GFP- puncta relative to control. Bafilomycin A₁ treated cells in contrast had a significantly lower percentage of RFP only puncta *p < 0.05, **p < 0.01.