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. 2022 Jan 24;12(4):1738–1755. doi: 10.7150/thno.64148

Figure 3.

Figure 3

CCZ1-MON1A was activated during autophagy and positively regulates autophagosome maturation. (A-B) GTP-RAB7 in autophagosomes isolated from normal and HBSS-treated N2a cells was determined by GST-R7BD affinity-isolation assay. (C) PIK3C3-associated GEF activity was increased in a starvation-induced autophagy condition. (D) Quantification of CCZ1-IPed PIK3C3 kinase activity in normal and HBSS-treated cells. (E-F) N2a cells were transiently transfected with GFP-CCZ1 and RFP-Lc3. Colocalization of CCZ1 and the LC3 under normal or HBSS-treated conditions were visualized under confocal microscope. Quantification data are presented as the mean ± SEM, n = 20-25 cells from 3 independent experiments. *P < 0.05, **P < 0.01, vs. the relative control. Scale bar: 5 μm. (G-H) N2a cells were transiently transfected with GFP-MON1A and RFP-Lc3. Colocalization of GFP-MON1A and the RFP-LC3 under normal or HBSS-treated conditions were visualized under confocal microscope. Quantification data are presented as the mean ± SEM, n = 20-25 cells from 3 independent experiments. *P < 0.05, **P < 0.01, vs. the relative control. Scale bar: 5 μm. (I-J) GTP-RAB7 in N2a cell was determined by GST-R7BD affinity-isolation assay. Data are quantified as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, vs. the relative control. (K-N) Autophagosome maturation in N2a and N2a cells over-expressing CCZ1-MON1A was determined by GFP-RFG-LC3 probe. Quantification data are presented as the mean ± SEM, n = 20-25 cells from 3 independent experiments. *P < 0.05, **P < 0.01, vs. the relative control. Scale bar: 5 μm. (O and P) SQSTM1 levels were determined by immunoblotting after over-expression of CCZ1-MON1A. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the relative control. (Q and R) SQSTM1 levels were determined by immunofluorescence after over-expression of CCZ1-MON1A. Quantification data are presented as the mean ± SEM, n = 20-25 cells from 3 independent experiments. *P < 0.05, **P < 0.01, vs. the relative control. Scale bar: 5 μm. (S and T) The N2a cells over-expressing CCZ1-MON1A were transfected with control SiRNA or Atg5 SiRNA. The levels of SQSTM1 were examined by immunoblotting. Data are quantified as mean ± SEM (n =3). *P < 0.05, **P < 0.01, vs. the relative control.