Figure 3. NOX4 plays a critical role in regulation of glycolysis by generating NAD⁺.

(a) OCR was determined in HPNE, HPNE/Kras^G12V and HPNE/Kras^G12V/shp16 cells. (b) Glucose uptake and lactate production, NAD⁺/NADH and NADP⁺/NADPH ratios were measured in HPNE, HPNE/Kras^G12V and HPNE/Kras^G12V/shp16 cells. (c) Immunoblotting analysis showed the NOX4 and p22^phox knockdown efficiency. Scrambled siRNA (sc) was used as a negative control here and in d–f. (d) NOX activity, NAD⁺/NADH, NADP⁺/NADPH ratios and glycolytic activity (glucose uptake, lactate production and ATP levels) were measured in NOX4-silenced HPNE/Kras^G12V/shp16 cells. (e) NOX activity, glucose uptake and lactate production levels were measured in p22^phox-silenced HPNE/Kras^G12V/shp16 cells. (f) Glucose uptake was measured in AsPc-1 and Panc-28 cells after siRNA depletion of NOX4 or p22^phox or co-expressing siRNA resistant NOX4 (NOX4-R). (g) The expression levels of NOX4 were analysed by qPCR and immunoblotting in NOX4-overexpressing HPNE cells. Data are presented as mean±s.d. (n=3). **P<0.01 for indicated comparison (Student unpaired t-test). (h) The pyruvate and lactate levels were measured in HPNE/NOX4 or HPNE/Kras^G12V/shp16 cells compared with parental HPNE cells using metabolite isotope tracing experiments with 13 carbon labelled glucose (U-¹³C₆ Glu). Data in b,d–f,h are presented as mean±s.d. (n=3). *P<0.05, **P<0.01 for indicated comparison (one-way analysis of variance (ANOVA) with the Newman Keul's multiple comparison test).