Extended Data Fig. 4: Autophagy is activated in C9 ALI-COs and astroglia.
a, Representative whole-section confocal microscopy slide-scanner images of control and C9 ALI-COs at 150 DIV, displaying autophagy marker P62+ (red), astroglial marker GFAP (green) and mature neuronal marker MAP2 (cyan) labeling. Quantification of p62+ area overlaps with either GFAP+ or MAP+ territories, expressed as mean ± s.e.m for correlation coefficients (CellProfiler); n = 3 independent ALI-COs per group; Two-tailed unpaired t-test. b, Representative confocal microscopy images of dissociated control/C9 ALI-CO cell cultures (±chloroquine [CQ] treatment) immunlabeled for P62/LC3 autophagy markers. Quantification of P62+ and LC3+ punctae in GFAP+ astroglia. Data expressed as mean ± s.d.; n = 25, 26, 28, 29 fields (4 cultures from 6 independent ALI-COs per group); Two-way ANOVA with Tukey’s posthoc test. c, Western blot (WB) of whole ALI-CO slice samples, showing autophagy marker LC3-immunolabelling with bands representing LC3-I early and LC3-II late autophagosomes. Schematic (right side) illustrates typical P62, LC3 level changes in authophagy activation vs failure. Scale bar=80 μm for a, 5 μm for b. See Supplementary Table 3 for detailed statistics and Source Data for unprocessed WB images.
