Journal: bioRxiv

Article Title: Activation of pro-survival autophagy by a small molecule promoting p62 oligomerisation

doi: 10.1101/2025.05.27.656309

Figure Lengend Snippet: A, Fluorescence microscopy images and quantification of mitophagy in Npc1 +/+ and Npc1 -/- MEFs expressing mt-mKeima cultured for 24 h in galactose medium supplemented with 50 nM rapamycin or 10 µM SQ-1. B , Fluorescence microscopy images and quantification of MitoSOX staining of Npc1 +/+ and Npc1 -/- MEFs cultured for 24 h in galactose medium supplemented with 50 nM rapamycin or 10 µM SQ-1. C , Phase-contrast images and cytotoxicity assay in Npc1 +/+ and Npc1 -/- MEFs cultured in galactose medium supplemented with 50 nM rapamycin or 10 µM SQ-1 for 72 h (phase contrast image) or 144 h (cytotoxicity assay). D , Cytotoxicity assay in control and NPC1 patient iPSC-derived cortical neurons after 4 weeks of neuronal differentiation, where neurons were treated with 50 nM rapamycin or 1-100 µM SQ-1 for 6 days. E , Fluorescence images and quantification of TUNEL + apoptotic nuclei in TUJ1 + cells treated as in D. F , Fluorescence images and quantification of LC3B and p62 puncta in TUJ1 + treated as in D. G , Schematic representation of the proposed mechanism by which SQ-1 sensitises p62 to mitochondria-generated ROS, leading to p62 oligomer formation, selective autophagy/mitophagy rescue, reduction in mitochondria with elevated ROS levels, and rescue of cell death. Data are mean ± SEM of n = 3 biological replicates. P values were calculated by one-way ANOVA followed by multiple comparisons with Dunnett’s test (A) or two-way ANOVA followed by multiple comparisons with Dunnett’s test (B, C, D). *P< 0.05; **P<0.01; ***P<0.001; ns (non-significant).

Article Snippet: The following primary antibodies were used: rabbit α-LC3B (NB100-2220, Novus Biologicals, 1:200), mouse α-p62 (610832, BD Biosciences, 1:200), mouse α-TUJ1 (4466, CST, 1:200).

Techniques: Fluorescence, Microscopy, Expressing, Cell Culture, Staining, Cytotoxicity Assay, Control, Derivative Assay, TUNEL Assay, Generated