Fig. 2
p300/CBP Inhibits Autophagic Flux in Neurons a Schematic diagram showing autophagic flux with changes in autophagic markers. b–e p300/CBP double knockout in primary mouse neurons reduces LC3 and p62 accumulation. b Representative immunoblots of LC3-I, −II, p62, and GAPDH in lysates of p300F/F/CBPF/F primary neurons infected lenti-ctrl or lenti-Cre. c–e Quantification of LC3-II (c), p62 (e) relative to GAPDH, and LC3-II/I (d), normalized to control. n = 6 wells from three independent experiments. *p < 0.05, ***p < 0.001 by unpaired t test. f Measuring autophagic flux by LC3 turnover assay. g, h p300/CBP double knockout in primary mouse neurons increases autophagic flux. g Representative immunoblots of LC3-I, −II and actin in lysates of p300/CBP double knockout neurons that were treated with BafA1 (10 nM) or DMSO for 24 h. h Quantification of autophagic flux based on the difference (increase) of LC3-II in response to BafA1 treatment, normalized to control (Cre-, BafA1-). n = 4 wells from two independent experiments. **p < 0.01 by unpaired t test. i–n CTB treatment in primary neurons reduces autophagic flux. i Representative immunoblot of LC3-I, −II, p62 and actin in lysates of primary mouse neurons treated with DMSO (control) or CTB (50 uM) for 24 h. j–l Quantification of acH3K18 relative to H3 (j), LC3-II (k) and p62 (l) relative to actin, normalized to control. n = 6 wells from four independent experiments. *p < 0.05, **p < 0.01 by unpaired t test. m Representative immunoblot of LC3-I, −II and actin in lysates of primary mouse neurons treated with DMSO (control), CTB (50 uM), BafA1 (10 nM), or CTB + BafA1 for 24 h. n Quantification of autophagic flux by the difference (increase) of LC3-II in response to BafA1, normalized to control (CTB-, BafA1-). n = 3 wells from three independent experiments. **p < 0.01 by unpaired t test. o Using mCherry-GFP-LC3 color change to measure autophagic flux. p Representative images of primary mouse neurons infected with lentivirus expressing mCherry-GFP-LC3 treated with CTB (50 uM) or DMSO (control) for 24 h. Scale bar: 10 μm. q Quantification of autophagic vesicles (AV). Autophagosomes (APG) are identified as yellow vesicles retaining both mCherry and GFP fluorescence. Autolysosomes (AL) are identified as red vesicles in which GFP fluorescence is quenched by the low pH in lysosomes. *p < 0.05, ***p < 0.001, two-way ANOVA, Tukey-Kramer post hoc analysis. r Ratio of the number of red vesicles to yellow vesicles per cell. **p < 0.01, unpaired t test. From two independent experiments, n = 12 cells (control), n = 17 cells (CTB). Values are mean ± SEM