Fig. 2

Knockdown of CMTM7 impairs autophagy. A Western blotting analysis of ATG12–ATG5 conjugation detecting by antibody against ATG12 in control and CMTM7 knockdown A549 cells. The intensity of the bands for ATG12–ATG5 conjugation protein was analyzed by ImageJ software and normalized to the grey density of β-actin. The average relative grey density with s.d. from three independent experiments was shown in (B) (**P < 0.01). C CMTM7 knockdown and control cells were treated with CQ (25 μM) or PBS as solvent control for 4 h. LC3B expression was detected by Western blotting, and β-actin was used as an internal standard. D Normalized densitometry data using ImageJ software according to the mean values of three independent experiments was presented as means ± s.d. (*P < 0.05, **P < 0.01). E Representative fluorescence microscopic images of endogenous LC3B co-localization with LysoTracker Red in CMTM7 knockdown and control cells. F The number of LC3B puncta co-localized with LysoTracker Red per cell was quantified and expressed as means ± s.d. of at least 20 cells (**P < 0.01). G Transmission electron microscopy analysis of autophagosome-like structures in CMTM7 knockdown and control cells. H The number of autophagic vacuoles per cell was quantified and expressed as means ± s.d. of at least 20 cells (**P < 0.01)