Fig. 1: TMEM189 negatively regulates the formation of autophagosome.

a HeLa cells were transfected with indicated siRNAs for 48 h, the mRNA and protein levels of TMEM189 were detected by RT-PCR and western blotting, respectively. b HeLa cells were transfected with indicated siRNAs for 48 h, and treated with or without BafA1 (10 nM) for the last 4 h. The endogenous LC3B dot distribution was analyzed by immunofluorescence and confocal microscopy. c Quantification of endogenous LC3B dots per cell was counted. Data are means ± SD of at least 50 cells scored. d HeLa cells were treated as (b). The levels of endogenous LC3B-II were detected by western blotting. e Quantification of amounts of LC3B-II relative to ACTB in cells. Average value in sicontrol-transfected cells without BafA₁ was normalized as 1. Data are means ± SD of results from 3 experiments. f HeLa cells were transfected with indicated siRNAs for 48 h, the levels of SQSTM1/P62 were analyzed by western blotting. g The stable GFP-LC3B-expressing HeLa cells were transfected with indicated siRNA-1/plasmids for 48 h, then incubated in the presence or absence of BafA1 (10 μM, 4 h). The GFP-LC3B dot distribution was observed by confocal microscopy. h Quantification of GFP-LC3B dots per cell was counted. Data are means ± SD of at least 50 cells scored. i The GFP-DFCP1-expressing HeLa cells were transfected with indicated siRNAs for 48 h, then incubated with or without EBSS for the last 1 h. The GFP-DFCP1 dot distribution was observed by confocal microscopy. j Quantification of GFP-DFCP1 dots per cell was counted. Data are means ± SD of at least 50 cells. *P < 0.05, **P < 0.01.