Fig. 2

The impact of Nestin on cellular antioxidant activity is mediated by the Nrf2–ARE pathway. a Immunoblotting was performed to detect the expression levels of Nestin and Nrf2 in NSCLC cells with or without Nestin knockdown. b Immunostaining was performed on sections of the xenograft tumors using the Tet-On 3G doxycycline-inducible gene expression system. The xenograft tumor samples were labeled with anti-Nrf2 (red), anti-Nestin (green), and DAPI (blue). Scale bar: 50 μm. c A luciferase assay was used to detect reporter gene activity from the AREs. NSCLC cells with or without Nestin knockdown were transiently transfected with an ARE luciferase reporter plasmid. At 24 h post-transfection, the cells were assayed for luciferase activity. The results are expressed as the fold-change of luciferase activity with respect to that of the vector control. d Western blot analysis representing the protein levels of NQO1, HO-1, and GCLM in NSCLC cells with or without Nestin knockdown. e The basal and induced protein levels of Nrf2 were lower in A549 cells with Nestin knockdown. NSCLC cells were treated with 100 μM tBHQ or 20 μM SF for 16 h to activate Nrf2. f–h The mRNA levels of Nrf2-downstream genes were analyzed by qPCR in Nestin-knockdown A549 cells. A549 cells were treated with DMSO (as a control), 100 μM tBHQ, or 20 μM SF for 16 h. i Nrf2-knockdown NSCLC cells transfected with vector or shNestin plasmids were transfected with the ARE luciferase reporter. At 24 h post-transfection, the cells were assayed for luciferase activity. j Nrf2-knockdown A549 cells transfected with vector or shNestin plasmids were treated with 200 μM H₂O₂ for 6 h, and flow cytometric detection of apoptosis was performed using Annexin V-FITC and PI. k Statistical analysis of the total apoptosis rates of the A549 cells described in j. The total apoptosis rates were calculated as the sum of the early and late apoptosis rates. Data are presented as the means ± SD of three independent experiments. **P < 0.01, N.S. represents no significant, Student’s t-test. Source data are available as a Source Data file