Fig. 7.

RAB37-associated autophagy flux. a, b RAB37 knockdown inhibits and over-expression promotes LC3B-II formation. Bafilomycin A₁ treatment results in protein accumulation of LC3B-II and SQSTM1. RAB37 overexpression, RAB37 knockdown and control HeLa cells were cultured and then starved in EBSS with or without bafilomycin A₁ (100 nM) for 1 h. The cell lysates were analysed by immunoblotting with antibodies as indicated. GAPDH was used as an endogenous control. Bottom: western blots were quantified for LC3B-II /GAPDH ratio and the SQSTM1/GAPDH ratio. Data are presented as means ± S.D. * stands for P < 0.05, ** stands for P < 0.01 (n = 3 independent experiments). c Autophagy flux revealed by the mCherry-GFP-LC3 tandem reporter in RAB37 knockdown (miR-RAB37 2#), over-expressed (stably expressing RAB37) and wild-type HeLa cell lines. Representative images of the cells transfected with the mCherry-GFP-LC3 reporter and grown in normal (control), EBSS medium (1 h) or EBSS with bafilomycin A₁ (100 nM) addition (1 h) respectively. Single channel (red, green or blue) and merged images were taken by confocal microscopy. d Statistic analysis of vesicles positive for both GFP and mCherry (autophagosomes) and for mCherry (autolysosomes) (>15 cells per experiment) in (c). Percentages of co-localized dots (mCherry-GFP-LC3 (yellow) / mCherry -LC3 (red)) were counted. Data are presented as means ± S.D. * stands for P < 0.05, (n = 3 independent experiments)