Figure 1.

p62 Is Essential for Neuronal Differentiation

(A–C) (A) Representative western blot analysis of p62 in (top) Co1^NES, Pat1^NES, and Pat2^NES cells and (bottom) CCo1^NES, KO1^NES, KO2^NES, and Pat1^NES cells. Tubulin was used as a loading control. n = 3 (n is defined as independent experiments). (B and C) Representative fluorescence images of Co1^NEU and Pat1^NEU after (B) 6 days differentiation, immunostained against α-tubulin (red) and DAPI (blue) (scale bar, 50 μm; n = 5), or (C) 20 days differentiation, immunostained against neurofilament (green), MAP2A/B (red), and DAPI (blue) or against β-III tubulin (green), HuC/D (red), and DAPI (blue) (scale bar, 100 μm; n = 5).

(D–F) Thirty neuronal bodies of Co1^NEU (black), Co2^NEU (white), and Co3^NEU (gray) (pooled under the name Co^NEU); Pat1^NEU (red); Pat1^NEU + p62 (brown); KO1^NEU (yellow); and Pat2^NEU (orange) were analyzed for (D) neuronal body area, (E) number of neuronal processes per neuronal body, and (F) width of neuronal processes. Data are expressed as mean ± SD and differences were tested by a two-tailed t test. ^∗∗∗p < 0.001, ^∗∗p < 0.01; n = 3.

(G) Linear Sholl analysis representation of 20-days-differentiated control neurons (pooled data from Co1^NEU, Co2^NEU, and Co3^NEU, under the name Co^NEU, dark gray); Pat1^NEU (red); and Pat2^NEU (orange). n = 3. Neuronal processes were analyzed from 15 neuronal bodies per line.

(H) Representative western blot analysis of steady-state levels of MAP2A/B, β-III tubulin, HuC/D, SOX2, and p62 in CCo1^NES and KO1^NES cells undifferentiated and after 14 or 50 days of differentiation. GAPDH was used as a loading control. n = 3.

See also Figures S1 and S2.