Figure 3.

Compromised Neuronal Differentiation due to Deficient p62 Can Be Rescued by NAC

(A) Quantification of the average CM-H₂DCFDA signal in four control NESCs (Co1^NEU, Co2^NEU, Co3^NEU, CCo1^NEU [pooled as Co^NEU]), Pat1^NES, Pat2^NES, and KO1^NES.

(B) As in (A), but on cells after 4 days of differentiation. Data are expressed as mean ± SD and differences were tested by a two-tailed t test. ^∗p < 0.05, ^∗∗p < 0.01; n = 3 with 50 cells analyzed per experiment.

(C) Cell viability assay of CCo1^NES, Co1^NES, Co2^NES, and Co3^NES (dark gray, pooled as Co^NES), Pat1^NES (red), and Pat2^NES (orange) after a 15-h treatment with 20 μM CCCP or 20 μM CCCP and 1 mM NAC as indicated. Data are presented as mean ± SD and differences were tested by a two-tailed t test. ^∗∗p < 0.01 and ^∗∗∗p < 0.001, n = 3.

(D) Representative bright-field images of Co1^NES and Pat1^NES cells, treated for 15 h with 20 μM CCCP or 20 μM CCCP +1 mM NAC. Scale bar, 100 μm; n = 3.

(E) Representative bright-field images of KO1^NES (±1 mM NAC) subjected to differentiation for 6 or 14 days. Scale bar, 100 μm; n = 3.

(F) Representative western blot analysis of Nrf2, KEAP1, and p62 in Co1^NES and Pat1^NES cells untreated and treated for 12 h with 10 μM CCCP or 10 μM oligomycin and 1 μM antimycin (O/A) as indicated. Tubulin was used as a loading control. n = 3.

(G–I) qRT-PCR analysis in Co1^NES (black) and Pat1^NES (red) cells, treated (striped) or untreated (solid) for 6 h with 10 μM CCCP and/or 1 mM NAC as indicated for (G) NRF2, GCLM, and xCT (left) and GSS, NQO1, and GSTM4 (right) (n = 4); (H) p62 (n = 3); or (I) NRF2, GCLM, and xCT (n = 3). β-actin was used as an endogenous control. Data are expressed as mean ± SD and differences were tested by a two-tailed t test. ^∗p < 0.05 and ^∗∗p < 0.01.

(J and K) qRT-PCR analysis of CCo1^NES (gray), KO1^NES (yellow), and Pat1^NES (red) cells for (J) GPX1, GPX4, GSTK1, GSTA1, and GSTP1 or (K) NQO1 and GSTM4 steady-state levels. β-actin was used as an endogenous control. Data are expressed as mean ± SD and differences were tested by a two-tailed t test. ^∗p < 0.05, ^∗∗p < 0.01, n = 3. See also Figure S4 and Table S2 for primers used.