Fig. 2: DOPAL-induced Nrf2 pathway activation and oxidative stress may contribute to p62 buildup.

A Schematic representation of Nrf2 activation by oxidative stress and DMF. The (i) direct binding of fumarate derivatives as well as (ii) cysteines oxidation following oxidative stress promote Keap1 detachment from Nrf2, which then translocates in the nucleus and binds to ARE. Real-time PCR of B SQSTM1 and C NRF2 mRNA levels in untreated and 100 µM DOPAL for 18 h. Data are pooled together from three independent experiments normalized to each untreated sample, and analyzed by one-sample t test (*P < 0.05, **P < 0.01). D Immunoblot of Nrf2 levels in BE(2)-M17 cells after treatments with 100 µM DOPAL, 10 µM H₂O₂ and 10 µM DMF for 18 h, and the untreated control. E Relative quantification after normalization to Actin used as loading control. Data are pooled together from three independent experiments normalized to each untreated sample, and analyzed by one-sample t test (*P < 0.05, ***P < 0.001). F Immunoblot of p62 in the same conditions and relative quantification of G p62 monomer and H p62 oligomers after normalization to Actin used as loading control. Data are pooled together from six independent experiments normalized to each untreated sample, and analyzed by one-sample t test (*P < 0.05, **P < 0.01). I Schematic representation of the Nrf2-ARE reporter. In transfected cells, the BFP is constitutively expressed while GFP expression depends on Nrf2 activation and binding to the 8xARE sequence. J Comparison of the molecular structures of dopamine and DOPAL. Both molecules have the catechol moiety (in purple) but only DOPAL has the aldehyde (in orange). K Representative confocal images of BE(2)-M17 cells transfected with Nrf2-ARE reporter and treated with 100 µM DOPAL, 100 µM Dopamine and 10 µM DMF for 18 h, as well as untreated cells. The constitutively expressed BFP (cyan) and the GFP (yellow) expressed under the activation of Nrf2 in the transfected cells are displayed as an overlay with the immunostaining for the endogenous p62 (magenta). Relative quantification of L Nrf2 pathway activation expressed as BFP/GFP fluorescence/cell, M total p62/cell measured as mean fluorescence intensity, N number of p62 puncta/cell. Data are collected form three independent experiments, normalized to the untreated samples, and analyzed by Kruskall–Wallis test with Dunn’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). O In the same experiment, principal component analysis of the three variables quantifies for each cell, where vectors (L, M, N) correspond to each quantification. P Molecular structures of antioxidants molecules N-Acetyl-cysteine (NAC), Ascorbic acid (ASC) and glutathione (GSH) used to prevent oxidative stress. Q Immunoblot with the anti-p62 antibody in the lysates of BE(2)-M17 treated for 18 h with 100 µM DOPAL, 100 µM Dopamine and the untreated cells. Prior to catechols administration, cells were pre-treated with 10 mM NAC, 250 µM ASC or 3 mM GSH for 1 h and then washed out. Quantification of R p62 monomer and S p62 oligomers in the lysates (after normalization to Actin used as loading control) of BE(2)-M17 treated with DOPAL or dopamine after the exposure to each antioxidant molecules. Data are pooled together from four independent experiments normalized to each untreated sample, and analyzed by one-sample t test (*P < 0.05, ***P < 0.001) and Mann–Whitney test (^$P < 0.05, ^$$P < 0.001, ^$$$P < 0.0001). A, I Artwork created with BioRender.com.