Fig. 3
Lysosomal CYPs and NOX2 activation contribute to ROS production. a IL-4 conditioned BMDMs were treated with MTX or MTX-MPs for 24 h, and gp91phox expression was analyzed by real-time PCR (left) and western blot (right). b, c IL-4 conditioned BMDMs were pretreated with 5 μM DPI for 30 min (b) or BMDMs were transfected with Cybb siRNAs for 12 h and stimulated with IL-4 for 12 h (c), then treated with MTX or MTX-MPs. ROS level and MFI of LysoSensor were detected by flow cytometry, Nos2, Tnf and Il6 expression was determined by real-time PCR. d, e The same as (a), except that Rac1 and Rac2 expression was analyzed by real-time PCR (left), Rac2 protein level (middle) and Rac2 activity (right) were analyzed by western blot, the location of Lamp1 (green) and Rac2 (red) was observed under a two-photon confocal microscope. Scale bar, 10 μm. f The same as (b), except that IL-4 conditioned BMDMs were pretreated with 10 μM EHT1864 for 30 min. g IL-4 conditioned BMDMs were treated with MTX or MTX-MPs for 4 h, and Cyp subunits expression was analyzed by real-time PCR. h–j IL-4 conditioned BMDMs were pretreated with 20 μM SKF-525A for 30 min and treated with MTX or MTX-MPs for 4 h. ROS level was analyzed by flow cytometry (h), Rac2 activity was analyzed by western blot (i) and NOX2 activity was analyzed by ELISA (j). k IL-4 conditioned BMDMs were pretreated with EHT1864 or DPI for 30 min and then treated with MTX or MTX-MPs for 4 h. ROS level was detected by flow cytometry. l-n Cyp1a1- or Cyp2j6-overexpressing BMDMs were stimulated with IL-4 for 12 h and treated with MTX or MTX-MPs for 4 h, ROS level was analyzed by flow cytometry (l), Rac2 activity was analyzed by western blot (m) and NOX2 activity was analyzed by ELISA (n). Unless otherwise specified, n = 3 biologically independent experiments were performed. Data are presented as mean ± SEM. P values were calculated using one-way ANOVA. **P < 0.01, ***P < 0.001, ****P < 0.0001
