Fig. 8
General proteostasis failure in NM-producing cells. a Top, Induction of hTyr expression in differentiated neuroblastoma SH-SY5Y cells before (OFF) and after (ON) treatment with doxycycline. Bottom, quantification of intracellular NM optical density post-hTyr induction. *p < 0.05, compared to OFF and ON 1d (ANOVA on ranks; Dunn’s post-hoc test). Scale bar, 12.5 μm. b Electron micrographs of NM granules in hTyr-expressing cells (6d post-hTyr induction). Arrowhead, double-membrane; asterisk, lipid droplet. Scale bar, 0.5 μm. c Immunoblot levels of Lamp1 (left) and LC3 (right) post-hTyr induction. d Lysosomal proteolysis post-hTyr induction. e Ubiquitin-proteasome (UPS) activity post-hTyr induction. f Immunoblot levels of p62 post-hTyr induction. g Representative images (left) and quantification (right) of aSyn oligomers (red) in hTyr-expressing cells as detected by aSyn-proximity ligation assay. Hoechst, nuclei (blue). BF bright-field. Scale bar, 25 μm. h Mitochondrial oxygen consumption rate (OCR) post-hTyr induction. i ROS production post-hTyr induction. MFI mean fluorescence intensity. j Cellular metabolic activity post-hTyr induction. k Number of surviving cells post-hTyr induction. l Representative bright-field photomicrographs with superimposed nuclear Hoechst fluorescent staining (blue) of hTyr-expressing cells before (OFF) and after (ON, 1–8d) hTyr induction. For clarity purposes, cell contour in weakly or no melanized cells are highlighted in white. Scale bar, 12.5 μm. In all panels, values are mean ± SEM. In a, d, e and i, n = 3 independent experiments. In c, h, j and k, n = 4 independent experiments. In a (bottom), n = 499 (OFF), n = 155 (1d), n = 111 (3d), and n = 48 (6d) cells. In g, n = 153 (OFF), n = 201 (3d), and n = 135 (6d) cells. Immunoblot densitometry was normalized to β-actin expression levels. BF bright-field, KDa kilodaltons. Unstained NM appears as brown. In c–f, h, j, *p < 0.05, compared to OFF (one-way ANOVA; Student–Newman–Keuls post-hoc test). In g, *p < 0.05, compared to OFF; #p < 0.05, compared to ON 3d (ANOVA on ranks; Dunn’s post-hoc test). In i, *p < 0.05, compared to OFF; #p < 0.05, compared to ON 3d (one-way ANOVA; Student–Newman–Keuls post-hoc test). In k, *p < 0.05, compared to OFF (ANOVA on ranks; Dunn’s post-hoc test)
