Fig 1: Defects in autophagic flux caused by the loss of KDM3B. (A and B) LC3B flux was analyzed in WT and KDM3B KO K562 cells (A) or THP1 cells (B) in the absence or presence of BafA1 (100 nM, 2 h). Cells were cultured and then starved in EBSS (4 h) with or without BafA1. KDM3B KO inhibited LC3B-II formation. BafA1 treatment resulted in the accumulation of LC3B-II and SQSTM1. The LC3B-II/LC3B-I ratio is indicated. (C) Autophagy flux was revealed by the mCherry-GFP-LC3 tandem reporter. The plasmid was transfected in WT or KDM3B KO 293T cells. Representative images of the cells cultured in normal, EBSS medium (4 h) or EBSS with BafA1 (100 nM) addition (2 h), respectively. Moreover, the formation of autophagosome (yellow: mCherry-positive; GFP-positive) and autolysosome (red: mCherry-positive; GFP-negative) was examined. Scale bar, 10 µm. (D) Statistical analysis of vesicles positive for both GFP and mCherry (autophagosomes) and for mCherry (autolysosomes) (>20 cells per experiment) in (C) Data (mean ± SD) were analyzed with two-way ANOVA. ns, not significant; ****P<0.0001. KDM3B, histone lysine demethylase 3B; WT, wild-type; KO, knockout; BafA1, bafilomycin A1; EBSS, Earle's Balanced Salt Solution.