Journal of Cancer Prevention

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Fig. 2.

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Fig. 2. Heregulin-β1 up-regulates manganese superoxide dismutase (MnSOD) expression through activation of extracellular signal-regulated protein kinase (ERK) and protein kinase B (Akt), and activates NF-E2-related factor 2 (Nrf2). (A, B) MCF-7 cells were preincubated with U0126 (0, 0.1, 1, or 5 μM) or LY294002 (0, 1, 5, or 20 μM) and exposed to heregulin-β1 (50 ng/mL) for 8 hours. The protein from cell lysates was resolved by SDS-PAGE and analyzed by Western blot analysis to measure the phosphoryation of ERK (A) or Akt (B). (C, D) The effects of pharmacologic inhibitors of ERK and Akt on expression of MnSOD were assessed by Western blot analysis after exposure to heregulin-β1 for 24 hours. Treatment conditions and other detials are same as above. (E) MCF-7 cells were transfected transiently with dominant-negative (DN)-ERK or control plasmid (CEP). After transfected with each vectors for 12 hours using DOTAP reagent, MCF-7 cells were subjected to heregulin-β1 treatment for 24 h. The expression of MnSOD was monitored by Western blot analysis. (G) MCF-7 cells were transfected transiently with hemaglutinin-tagged full-length Akt (HA-Akt) or kinase-dead Akt (KD-Akt). After transfection with each vector for 12 h using DOTAP reagent, MCF-7 cells were treated with heregulin-β1 for additional 24 hours. Proteins from cell lysates were resolved by SDS-PAGE and analyzed by Western blot analysis. (H) Nuclear extracts from MCF-7 cells were prepared after treatment with heregulin-β1 (50 ng/mL) for indicated time periods. Immunoblots of nuclear lysates from treated MCF-7 cells were probed with the Nrf2 specific antibody. (I) Immunocytochemical analysis was performed using anti-Nrf2 antibody after the treatment of MCF-7 cells with 50 ng/mL of heregulin-β1 for 4 hours. Cells were stained with propidium iodide and analyzed by confocal microscopy. Scale bar, 50 μm. (J) Heregulin-β1 induces DNA binding activity of Nrf2 in MCF-7 cells. MCF-7 cells were treated with heregulin-β1 (50 ng/mL) for indicated time periods. The DNA-binding activity of Nrf2 in MCF-7 cells stimulated with heregulin-β1 was measured by electrophoretic mobility shift assay (EMSA). The nuclear extract isolated from heregulin-β1-treated cells was used for EMSA as described under Materials and Methods. (K) The heregulin-β1-mediated increase in the transcriptional activation of antioxidant response elements (ARE) was measured by the luciferase reporter gene assay. After overnight transfection, cells were exposed to 50 ng/mL heregulin-β1 for 8 hours, and treated with reporter lysis buffer for the measurement of the luciferase activity.
J Cancer Prev 2021;26:54~63 https://doi.org/10.15430/JCP.2021.26.1.54
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