Fig. 4

Effect of CHMP2A on autophagy in PC12 cells in vitro. a, b Representative Western blot and quantification of p62, LC3, and CHMP2A expression in PC12 cells infected with CHMP2A-specific shRNA or vector shRNA for 72 h. c Representative TEM analyses of autophagy in CHMP2A-silenced PC12 cells. (n = 6, Student’s t test. *P < 0.05, ***P < 0.001, ****P < 0.0001 vs. sh-vector group) d Autophagosome accumulation in CHMP2A-knockdown PC12 cells as shown by TEM. Red arrows indicate autophagosomes. Scale bar, 10 μm. e Representative RT-qPCR analyses of LC3 and p62 in PC12 cells transduced with CHMP2A or vector (20 MOI) (n = 3, Student’s t test. ****P < 0.0001 vs. sh-vector group). f PC12 cells were transfected with increasing doses of CHMP2A constructs (for 72 h) before OGD-Rep and were subjected to OGD or normoxia (Nor) as a control. Representative Western blot showing p62, LC3, and CHMP2A expression in PC12 cell lysates. g PC12 cells were transfected with CHMP2A constructs for 72 h and then were subjected to OGD-Rep. Cell viability was determined using the CCK-8 assay at 2 h (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). h Quantification of p62, LC3, and CHMP2A expression in PC12 cell lysates. i Quantitative analysis of LC3B puncta per cell (n > 25). j Representative immunofluorescence images of LC3 (red) and DAPI (blue) double-staining in PC12 cells transfected with Lenti-EGFP vector or Lenti-EGFP-CHMP2A virus before OGD-Rep. Puncta correspond to phagophores and/or autophagosomes. Scale bar, 50 μm. k Representative immunofluorescence images of p62 (red) and DAPI (blue) double-staining in PC12 cells transfected with Lenti-EGFP vector or Lenti-EGFP-CHMP2A virus before OGD-Rep. The data are expressed as means ± SEM (n = 3–6), one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001