Fig 2: Effect of ionizing radiation on nuclear translocation of Nrf-2 and NF-κB and expression of HO-1 and Mn-SOD.(A) Nuclear levels of Nrf-2 in control and irradiated (4 Gy) EL-4 cells were probed by EMSA in the presence and absence of U0126. (B) Representative flow cytometric histograms show intracellular levels of HO-1. The frequency of PE-HO-1+ cells (gate RN-1) is shown in (C). Bar graph shows percent HO1+ cells. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05, as compared to untreated cells. (D) Levels of HO-1 and Mn-SOD in cytosolic extracts were measured by western blotting. (E) Nuclear levels of NF-κB in control or irradiated (4 Gy) EL-4 cells were assessed by EMSA. IκBα degradation at corresponding time points was monitored by western blotting in cytosolic fractions.

Fig 3: ERK or Nrf-2 knockdown EL-4 cells are significantly more radiosensitive than wild type cells.(A) EL-4 cells transfected with scrambled shRNA or ERK or Nrf-2 shRNA plasmid were exposed to ionizing radiation (4 Gy) and cultured for 48 h. Apoptosis was estimated by propidium iodide staining and flow cytometry. (B) The frequency of apoptotic cells (gate RN1) is shown in the bar graph. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05, as compared to unirradiated cells and #p<0.05, as compared to irradiated cells. (C) Genomic DNA from wild type and knock down EL-4 cells cultured for 48 h post radiation exposure or alone was resolved on agarose gel and stained with ethidium bromide. DNA ladder indicates cells undergoing apoptosis.

Fig 4: Inhibitors of ERK or Nrf-2 significantly enhanced radiosensitivity of tumor cells.(A) EL-4 cells were incubated with different concentration of pharmacological inhibitors of ERK (10 µM for 2 h) or JNK (10 µM for 2 h) or P38 (10 µM for 2 h) or NF-kB inhibitory peptide (10 µM for 2 h) or Nrf-2 (ATRA) (5 µM for 2 h) or HO-1 (SnPP) (5 µM for 2 h) or TrxRd1 (auranofin) (25 nM for 2 h) or Ras (FTA) (10 µM for 2 h) and were cultured for 48 h in 5% CO2 at 37°C with or without exposure to 4 Gy ionizing radiation. Cell death was analyzed by propidium iodide staining and flow cytometry. Representative flow cytometric histograms show apoptotic population as pre-G1 cells. (B) Bar graph represent radiation induced apoptosis in EL-4 cells incubated in the presence of different inhibitors. Data points represent mean±S.E.M. from nine replicates from three independent experiments. *p<0.05, as compared to untreated cells and #p<0.05, as compared to irradiated cells (C) EL-4 cells incubated in the presence of different pharmacological inhibitors were assessed by clonogenic assay for clonogenicity. Bar graph shows number of colonies in different treatment groups. Each bar shows mean±S.E.M from nine replicates from three independent experiments. *p<0.01, as compared to untreated cells and #p<0.01, as compared to irradiated cells.

Fig 5: Schematic representation showing the involvement of ERK-Nrf-2 signaling pathway in radioresistance of EL-4 cells.