Fig. 6

Enhancing Autophagy Flux Mitigates p300-mediated Tau Secretion a Schematic diagram showing autophagic flux and predicted changes in autophagic markers with rapamycin (rapa) treatment. b–f Rapa treatment in primary neurons. b Representative immunoblots of LC3-I, LC3-II, p62 and actin in lysates of rat primary neurons treated with rapa (0.25 μM) or DMSO (ctrl) for 24 h. Quantification of levels of LC3-II/I (c) and p62 (d) relative to actin, normalized to control. e Quantification of intracellular tau levels by ELISA, normalized to control. f Quantification of tau secretion over 3 h by ELISA, normalized to intracellular tau levels and normalized to control. (b–f) n = 6 wells from three independent experiments. ***p < 0.001, **p < 0.01 by unpaired t test. g, h Rapamycin treatment in HEK293T cells with and without p300 overexpression. g Representative immunoblots of LC3-I, LC3-II, p62, total tau (t-tau) and GAPDH in lysate and conditioned medium of HEK293T cells transfected with tau alone or tau+p300. Serum-starved HEK293T cells were treated with DMSO (ctrl) or rapamycin (1 μM) for 24 h. h Quantification of t-tau secretion in (g) normalized to intracellular levels and normalized to control. n = 3 wells from three independent experiments. *, #p < 0.05 by one-way ANOVA and Sidak’s multiple comparisons test. i, j Primary neurons treated with CTB and rapa. i Representative immunoblots of LC3-I, LC3-II, p62, and actin in lysates of mouse primary neurons treated with DMSO (ctrl), CTB (50 μM), rapa (0.25 μM) and CTB + rapa. j Quantification of tau secretion over 3 h by ELISA of neurons as in (i), normalized to intracellular tau levels and normalized to control (CTB- rapa-). n = 6 wells from three independent experiments. *, #p < 0.05, **p < 0.01 by one-way ANOVA and Sidak’s multiple comparisons test. Values are mean ± SEM