Progressive Rotavirus Infection Downregulates Redox-Sensitive Transcription Factor Nrf2 and Nrf2-Driven Transcription Units

Figure 8

Neither translational arrest nor inhibited nuclear export of mRNA triggers downregulation of the Nrf2/HO-1 axis during RV infection. (a) Mock-infected and RV-SA11-infected MA104 cells were treated with C16 (0.5 μM and 1 μM) or vehicle control (DMSO) during final media addition (1 hpi). Protein levels of Nrf2, HO-1, p-eIF2α, and eIF2α were assessed in cellular extracts prepared at 9 hpi by immunoblot analyses. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, C16- (0.5 μM) treated mock-infected, and C16- (1 μM) treated mock-infected groups, respectively. (b) Mock-infected and RV-SA11-infected MA104 cells were treated with Hemin (5 μM; added at 3 hpi) and C16 (1 μM; added at 1 hpi)/DMSO (vehicle control). Protein levels of Nrf2, HO-1, p-eIF2α, and eIF2α were finally assessed in cellular extracts prepared at 9 hpi by immunoblot analyses. Relative fold changes of proteins are represented; “,” “#,” and “ns” represent comparisons with respect to vehicle-treated mock-infected, Hemin-treated mock-infected, and Hemin-treated infected groups not treated with C16, respectively. (c) MA104 cells transfected with empty vector (PLKO.1-TRC) or NSP3 shRNA for 24 hours were further mock infected or infected with RV-SA11. Cellular extracts prepared at 9 hpi were analyzed by SDS-PAGE/western blotting to assess protein levels of Nrf2, HO-1, and RV-NSP3. Relative fold changes of proteins are represented; “ns” and “” represent comparisons with respect to the PLKO.1-TRC transfected mock-infected control; “#” represents comparison with respect to the NSP3 shRNA transfected mock-infected group. (d) Hemin-treated (5 μM, added at 3 hpi) MA104 cells were either infected with RV-SA11 in the presence or absence of pretransfected NSP3 shRNA or kept mock infected. Cellular extracts prepared at 9 hpi were checked for protein levels of Nrf2, HO-1, and RV-NSP3 by SDS-PAGE/immunoblotting. Relative fold changes of proteins are represented; “,” “#,” and “ns” represent comparisons with respect to the Hemin-untreated mock-infected group transfected with PLKO.1-TRC, Hemin-treated mock-infected group, and Hemin-treated RV-SA11-infected group transfected with PLKO.1-TRC, respectively. (e) MA104 cells were treated with H₂O₂ (0.5 mM and 1 mM) for 2 hours. Levels of p-eIF2α, eIF2α (from whole cell extracts), Nrf2 (from whole cellular extracts as well as nuclear fractions), and PABPC1 (from nuclear fraction) were investigated by SDS-PAGE/immunoblotting. Relative fold change of Nrf2 is represented; “” represents comparison with respect to the H₂O₂-untreated control group.

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