Fig. 2: Protein aggregates accumulate in quiescent NSPC cultures despite the increase in autophagy machinery content.

a Experimental design. Hippocampal NSPC cultures are grown in the presence of fibroblast growth factor 2 (FGF2) and quiescence is induced by the addition of bone morphogenetic protein 4 (BMP4). Removing BMP4 reverses quiescence. b Quantification of protein aggregates measured by ProteoStat labeling at 4 and 8 days of quiescence induction and 4 days of quiescence reversion. At least 15 cells were analyzed per experiment and condition (n = 4 independent experiments). Statistics: one-sample t-test, two-tailed. c Representative confocal immunofluorescence images of Q-NSPC and A-NSPC cultures stained with ProteoStat (red). Scale bar: 25 μm. d Quantification of proteasome 20S chymotrypsin activity (Δ) in A- and Q-NSPCs upon its inhibition using 10 μM MG132 (n = 5 independent experiments for MG132 treated cells and n = 6 independent experiments for control untreated cells). Statistics: two-way ANOVA. e Quantification of LC3-II levels in A- and Q-NSPCs (n = 7 independent experiments). Statistics: one-sample t-test, two-tailed. f Immunoblots of LC3 in A- and Q-NSPCs after 100 nM BafA1 treatment (6 h). β-actin was used as a loading control. g The autophagy flux expressed as the ratio of LC3-II(+BafA1)/LC3-II(-BafA1) was equivalent for A-NSPCs and Q-NSPCs. h (Left) Quantification of TAX1BP1 protein level measured as fluorescence intensity. Each point represents individual cultures (n = 3 independent experiments). Statistics: one-sample t-test, two-tailed. (Right) Representative immunofluorescence confocal images of TAX1BP1 in A- and Q-NSPCs. Scale bar: 25 μm. i Quantification of p62 levels in A- and Q-NSPCs (n = 4 independent experiments). Statistics: one-sample t-test, two-tailed. j Immunoblots of p62 in A- and Q-NSPCs after 100 nM BafA1 treatment (6 h). β-actin was used as a loading control. k The autophagy flux expressed as the ratio of p62(+BafA1)/p62(-BafA1) was equivalent for A-NSPCs and Q-NSPCs. l (Left) Quantification of LAMP2 protein level measured as fluorescence intensity. Each point represents individual cultures (n = 6 independent experiments). Statistics: one-sample t-test, two-tailed. (Right) Representative immunofluorescence confocal images of LAMP2 in A- and Q-NSPCs. Scale bar: 10 μm. m (Right) In electroporated cells expressing the pRFP-GFP-LC3 sensor, autophagosomes display GFP and RFP fluorescence (yellow puncta), whereas autolysosomes display RFP fluorescence only, because GFP is denatured at the acidic pH of the lysosome. (Left) Quantification of the number of autophagosome puncta (GFP⁺RFP⁺) in A- and Q-NSPCs after 100 nM BafA1 treatment (6 h) (n = 3 independent experiments). Statistics: two-way ANOVA. Data in panels (b, d, e, g, h, i, k, l, and m) are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Representations shown in (a and n) were created with BioRender.com. Source data and all blots are provided as a Source Data file. A, active NSPCs. Q, quiescent NSPCs. Qrev quiescence-reverted NSPCs.