Fig. 4: NDP52^GE degrades P301S-TAU via autophagy and is more efficient than NDP52^WT.

A WB analysis of human SH-SY5Y P301S-TAU inducible cells non-treated (TET −) or treated (TET +) with tetracycline for 24 h to induce P301S-TAU expression. To block the autophagosome–lysosome fusion, cells were treated with NH₄Cl (20 mM, 24 h). Images are representative of three independent experiments. Signals from LC3II, TAU, and NDP52 antibodies were measured and normalized to the corresponding signal of VINCULIN. Data are reported as means ± SEM. (two-tailed unpaired t-test). B WB analysis of human SH-SY5Y P301S-TAU inducible cells overexpressing GFP, GFP-NDP52^WT or GFP-NDP52^GE and treated with tetracycline to induce P301S-TAU expression. After 16 h, the medium was substituted with a TET-free medium for 8 h in order to evaluate and compare TAU degradation rate. Data are reported as means ± SEM. (two-tailed unpaired t-test). C Representative confocal images of human SH-SY5Y P301S-TAU inducible cells non-treated (TET −) or treated (TET +) with tetracycline for 24 h to induce P301S-TAU expression, fixed and stained with anti-TAU (magenta staining) antibody and with DAPI (blue staining in the merge panels) to detect nuclei. Intensity of anti-TAU signal was measured, and results are reported in the graph. Images are representative of three independent experiments and more than 70 cells for each condition were analyzed. (two-tailed unpaired t-test). Scale bar: 10 μm. D Representative confocal images of human SH-SY5Y P301S-TAU inducible cells overexpressing GFP-NDP52^WT or GFP-NDP52^GE and treated as in (B). Cells were left untreated (ctrl) or were treated with the autophagic inhibitor 3-Methyladenine (+3-MA) for 8 h. Cells were fixed and stained with anti-TAU antibody and with DAPI to detect nuclei. Intensity of anti-TAU signal was measured, and results are reported in the graph. Images are representative of three independent experiments and more than 200 cells for each condition were analyzed. (two-tailed unpaired t-test).