Fig. 4

CMTM7 increases the activity of ATG14L-linked VPS34 complex. A HeLa cells were co-transfected with GFP-CMTM7 and mCherry-WIPI-1 plasmids, and after transfection for 24 h, the representative confocal microscopy images of mCherry-WIPI-1 distribution were shown. Scale bars: 5 µm. B mCherry-WIPI-1 puncta per cell were quantified by ImageJ software and normalized to the background of mCherry diffusion, and expressed as means ± s.d. of at least 20 cells (**P < 0.01). C Endogenous ATG14L was immunoprecipitated by control and GFP-CMTM7-overexpressing HeLa cells, and ATG14L-linked VPS34 kinase activity was measured by using the Class III PI3K ELISA Kit (*P < 0.05). D Western blotting analysis of LC3B expression in CMTM7 overexpressing and control cells treated with CQ (25 μM) for the final 4 h and/or 3-MA (10 mM) for the final 6 h was performed. β-actin was used as an internal standard. The intensity of the bands for LCB-II 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 (E) (***P < 0.001; ns, not significant). F GFP-LC3B HeLa cells were transfected with empty vector or CMTM7 plasmid and treated as described in (D). Representative confocal microscopy images of GFP-LC3B distribution were presented. G GFP-LC3B puncta per cell were quantified and expressed as means ± s.d. of at least 20 cells (*P < 0.05; ns, not significant). H Control and CMTM7-overexpressing cells were transfected with control siRNA (si-NC) and siVPS34 (si-VPS34). Western blotting analysis of the levels of VPS34 and LC3B. β-actin was used as an internal standard