Fig. 3

Knockdown or pharmacological inhibition of G6PD attenuated LPS-elicited ROS production. a Microglia-enriched cultures grown in culture chambers were transfected with 10 nM scramble siRNA (SS) or G6PD siRNAs (GS1 and GS2), and 30 h later, the cultures were treated with LPS. Superoxide production in microglia-enriched cultures was measured by SOD-inhibitable WST-1 reduction. b G6PD inhibitors 6-AN and DHEA attenuated superoxide production in microglia-enriched cultures. c Rat mixed-glia cultures grown in black 96-well plates were pretreated with vehicle, 6-AN, or DHEA at the indicated concentration for 30 min prior to the addition of LPS in phenol red-free medium. Fluorescent probe H₂DCFDA was used to detect inhibition of 6-AN or DHEA on iROS production at 6 h after LPS addition. d, e At 2 days after LPS treatment, iROS were detected by fluorescent probe CM-H₂DCFDA (d) and quantification of the fluorescent intensity (e). f, g At 2 days after LPS treatment, CM-H₂DCFDA (green) or anti-4-HNE antibody (red) detected decreases in oxidative stress in microglia-enriched cultures pretreated with 10 μM 6-AN and 100 μM DHEA. Results are mean ± SEM of three to four experiments performed in triplicate (a–c, e, f). *p < 0.05 compared with vehicle-treated controls. ^#p < 0.05 compared with LPS-treated cultures