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

Figure 5

Depletion of the Nrf2/HO-1 axis beyond the early hours of RV-SA11 infection is independent of redox regulation and Nrf2 posttranslational modifications. (a) Mock-infected and RV-SA11-infected MA104 cells were treated with sodium arsenite (NaAsO₂; 25 μM and 50 μM) or vehicle control (H₂O) for 2 hours (added at 7 hpi) before cellular extract preparation at 9 hpi. Protein levels of Nrf2 and HO-1 were finally studied by SDS-PAGE/immunoblotting. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, NaAsO₂- (50 μM) treated mock-infected, and NaAsO₂- (25 μM) treated mock-infected groups, respectively. (b–e) Mock-infected and RV-SA11-infected MA104 cells were treated with PMA (0.1 μM and 0.2 μM) or vehicle control (ethanol) at the time of final media addition (at 1 hpi). (b) Cellular extracts were prepared at 9 hpi for assessing protein levels of Nrf2, pNrf2 (Ser40), and HO-1 by immunoblot studies. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, PMA- (0.1 μM) treated mock-infected, and PMA- (0.2 μM) treated mock-infected groups, respectively. (c) Cells fixed at 9 hpi were processed for confocal microscopy to visualize Nrf2. Scale bar, 20 μM. (d) Nrf2 CTCF from each panel of (c) was shown. “” and “#” represent comparisons with respect to vehicle-treated mock-infected and PMA- (0.2 μM) treated mock-infected groups, respectively. (e) Quotients of nuclear hollowing (NH_Q) of Nrf2 in PMA-treated+mock-infected and PMA-treated+RV-SA11-infected cells are represented with respect to the vehicle- (ethanol) treated+mock-infected control. (f) Mock-infected and RV-SA11-infected MA104 cells were treated with TSA (0.2 and 0.4 μM, respectively) or vehicle control (DMSO) (added during final media addition). Cellular lysates prepared at 9 hpi were immunoprecipitated with anti-Nrf2 antibody, and levels of acetyl lysine were checked in the immunoprecipitate by immuonblotting. Levels of HO-1 (from unfractionated input lysates) and Nrf2 (from nuclear extracts) were assessed simultaneously. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, TSA- (0.4 μM) treated mock-infected, and TSA- (0.2 μM) treated mock-infected groups, respectively. (g) Mock-infected and RV-SA11-infected MA104 cells were treated with Tunicamycin (TM; 5 μM and 10 μM, respectively) or vehicle control (DMSO) 2 hours before cellular extract preparation at 9 hpi for assessing protein levels of Nrf2, HO-1, p-PERK, and PERK by immunoblot analyses. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, TM- (5 μM) treated mock-infected, and TM- (10 μM) treated mock-infected groups, respectively.

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