Figure 6
Pentose phosphate pathway activity is increased upon p62 deficiency. (A,B) NAD(P)H levels were obtained through the analysis of NADH autofluorescence. The NAD(P)H values were calculated by subtracting the background from the minimum fluorescence after addition of FCCP (1 μM) which oxidises all the mitochondrial NADH minimising the NADH fluorescence (see Fig. 2B). NAD(P)H levels from untransfected, SCR and p62 KD SH-SY5Y cells (A) and control and p62-deficient fibroblasts (B) representing the mean of at least 3 independent experiments ±SEM. * indicates p < 0.05 and *** indicates p < 0.001 compared with the values in control cells. (C–E) GSH levels were analyzed by measuring the monochlorobimane (MCB) fluorescence. Representative images showing the cell fluorescence after MCB incubation (50 μM) in fibroblasts from control 2 (C2) and patient 2 carrying the K238del mutation (C). Scale bar represents 44 μm. The GSH levels were obtained after evaluation of the MCB fluorescence in fibroblasts from all p62 mutant carriers and the obtained values were compared to those from the control fibroblasts before (D) and after digitonin treatment (40 nM) (E). All data represents the mean of at least 3 independent experiments ±SEM. In all cases * indicates p < 0.05 and ** indicates p < 0.01 compared with the values in control cells.
