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

Figure 9

RV-mediated attenuation of Nrf2 is sensitive to proteasome inhibition and associated with increased K48-linked ubiquitination. (a, b) Mock-infected and RV-SA11-infected MA104 cells were treated with MG132 (5 μM)/vehicle control (DMSO) during final media addition (1 hpi) before harvesting at (a) 6 hpi and (b) 9 hpi. Protein levels of Nrf2, HO-1, and IRF3 were subsequently analyzed from cellular extracts by SDS-PAGE/immunoblotting. Relative fold changes of proteins are represented; “” and “ns” represent comparisons with respect to vehicle-treated mock-infected and MG132- (5 μM) treated mock-infected groups, respectively. (c) Lysates from mock-infected and RV-SA11-infected (9 hpi) MA104 cells cotreated with MLN4924 (0.5 μM and 1 μM; added at 1 hpi) or vehicle control (DMSO) were subjected to SDS-PAGE/immunoblotting for analyzing protein levels of Nrf2 and HO-1 and Cullin3. The neddylated form of Cullin3 is marked by an arrow. Relative fold changes of proteins are represented; “,” “#,” and “$” represent comparisons with respect to vehicle-treated mock-infected, MLN4924- (1 μM) treated mock-infected, and MLN4924- (0.5 μM) treated mock-infected groups, respectively. (d–f) Mock-infected and RV-SA11-infected MA104 cells were treated with MLN4924 (1 μM) or vehicle control (DMSO) at the time of final media addition. Cells fixed at 9 hpi were processed for (d) confocal microscopy to visualize Nrf2. Scale bar, 20 μM. (e) Nrf2 CTCF from each panel of (d) is shown. “” and “#” represent comparisons with respect to vehicle-treated mock-infected and MLN4924- (1 μM) treated mock-infected groups, respectively. (f) Quotients of nuclear hollowing (NH_Q) of Nrf2 in MLN4924-treated+mock-infected and MLN4924-treated+RV-SA11-infected cells were represented with respect to vehicle- (DMSO-) treated+mock-infected control. (g) Lysates from mock- and RV-SA11-infected (3 and 9 hours) MA104 cells were immunoprecipitated with anti-Nrf2 antibody. Similarly, lysates from MLN4924- (0.5 μM) treated RV-SA11-infected (9 hours) and mock-infected MA104 cells were immunoprecipitated with anti-Nrf2 antibody. Immunoprecipitates were subjected to SDS-PAGE/immunoblotting with anti-K48-linked Ub antibody. The presence of Nrf2 was evaluated in input lysates. Relative fold change of K48-linked ubiquitinated Nrf2 was assessed after normalization with respective input lanes. “” and “#” represent comparisons with respect to MLN4924-untreated mock-infected and MLN4924-treated mock-infected groups, respectively. (h) Lysates from mock- and RV-SA11-infected (3 and 9 hours) MA104 cells were immunoprecipitated with the anti-K48-Ub antibody. Similarly, lysates from MLN4924- (0.5 μM) treated RV-SA11-infected (9 hours) and mock-infected MA104 cells were immunoprecipitated with the anti-K48-Ub antibody. Immunoprecipitates were subjected to SDS-PAGE/immunoblotting with the anti-Nrf2 antibody. The presence of Nrf2 was evaluated in input lysates. Relative fold change of K48-linked ubiquitinated Nrf2 was assessed after normalization with respective input lanes. “” and “#” represent comparisons with respect to MLN4924-untreated mock-infected and MLN4924-treated mock-infected groups, respectively. (i, j) Lysates from MLN4924- (0.5 μM) treated RV-SA11-infected (9 hours) and mock-infected MA104 cells were immunoprecipitated with (i) anti-Nrf2 or (j) anti-K48-Ub antibody. Similarly, lysates from mock- and RV-SA11-infected (3 and 9 hours) MA104 cells were immunoprecipitated with (i) anti-Nrf2 or (j) anti-K48-Ub antibody. The amount of cellular lysates which were subjected to immunoprecipitation (to assess K48-linked ubiquitinated Nrf2) was normalized on the basis of prior normalization of Nrf2 input levels such that Nrf2 levels remain the same in each input lane. Immunoprecipitates were subjected to SDS-PAGE/immunoblotting and further probed with (i) anti-K48-Ub antibody and (j) anti-Nrf2 antibodies. (k, l) Mock-infected and RV-SA11-infected MA104 cells were treated with MG132 (5 μM) during final media addition (1 hpi) before harvesting at 9 hpi. Cellular lysates were immunoprecipitated with (k) anti-Nrf2 antibody or (l) anti-K48-Ub antibody. Immunoprecipitates were finally run on SDS-PAGE, transferred on to a PVDF membrane, and probed with (k) anti-K48-Ub or (l) anti-Nrf2 antibody. Relative fold change of K48-linked ubiquitinated Nrf2 was assessed after normalization with respective input lanes; “” represents comparison with respect to the MG132-treated mock-infected control.

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