Fig. 2

IκBα silencing modulates sensitivity to the DNA-damaging agent cisplatin and induces metabolic rewiring and chronic oxidative stress. a Immunoblotting of IκBα and vinculin in A549 cells infected with empty vector (shCTR) and two independent sh for IκBα (shIΚBD2 and shIΚBD7). b Luciferase assays of NF-κB activity in A549 cells infected with empty vector (shCTR) and two independent shIκBα (shIKBD2 and shIKBD7). Cells were transfected with NF-κB luciferase reporter or a control vector. Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. **P < 0.01; ***P < 0.001. c Percentage of apoptotic A549 cells previously described in (a) treated with 5 μM and 10 μM cisplatin for 48 h, assessed by Annexin V. Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. *P < 0.05; **P < 0.01. d Growth curves of A549 described in (a) treated with 5 μM and 10 μM cisplatin. Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. *P < 0.05. e Gene set enrichment analysis plot of NF-κB signaling, apoptotic hallmarks (f) oxidative phosphorylation (g) and reactive-oxygen-species pathway (h). IκBα negatively correlated in A549 cells described in (a). i Quantification of mitochondria content by FACS analysis in A549 cells described in (a). Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. *P < 0.05; **P < 0.01. j Representative Western blot showing five ETC proteins (ATP5A, ATP synthase, H+ transporting, mitochondrial F1 complex, α subunit; UQCR2, ubiquinol-cytochrome c reductase core protein II; SDHB, succinate dehydrogenase complex iron sulfur subunit B; COXII, mitochondrially encoded cytochrome c oxidase II; NDUFB8, NADH:ubiquinone oxidoreductase subunit B8) in A549 cells. I, II, III, IV, and V indicate ETC complexes. Vinculin is provided as a loading control. k A549 cells described in (a), were analyzed for ETC complexes activity, lactate production (l). Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. *P < 0.05; **P < 0.01; ***P < 0.001. m Flow cytometry experiments measuring intracellular ROS levels in A549 cells previously described in (a). Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. **P < 0.01; ****P < 0.0001. n A549 cells previously described in (a) were analyzed for Lipoperoxidation and cholesterol esters production (o). Data are shown as mean ± sem (n ≥ 3 independent experiments). P-values are from Student’s t-test. **P < 0.01; ***P < 0.001