(a) NRBF2 KO MEFs display impaired autophagosome acidification. WT and NRBF2 KO MEFs are transiently transfected with mCherry-GFP-LC3B. WT MEFs expressing mCherry-GFP-LC3B contain many red-only puncta along with yellow (presence of both red and green) puncta. The number of both red-only puncta and yellow puncta markedly increases after rapamycin (Rap; 50 μg ml−1, 4 h) or starvation (HBSS, 4 h) treatment, suggesting the increased autophagolysosomes and nascent autophagosomes. In contrast, NRBF2 KO MEFs expressing mCherry-GFP-LC3B contain quite fewer red-only puncta and yellow puncta, even after Rap or starvation treatment. (b) The quantification results show that both the numbers of red-only puncta and percentage of red-only puncta (verses total puncta) in KO MEFs were markedly reduced. (Data are shown as mean±s.e.m., P values are indicated on the figures, Mann-Whitney U-test, n=20 randomly selected cells, a representative result from three independent repeats), indicating both the autophagolysome number and autophagosome acidification rate are reduced in KO cells. Scale bars, 20 μm or 10 μm (amplified regions). 