Synergism and Antagonism of Two Distinct, but Confused, Nrf1 Factors in Integral Regulation of the Nuclear-to-Mitochondrial Respiratory and Antioxidant Transcription Networks

Figure 5

A negative effect of α-Pal^NRF1 on Nfe2l1, Nfe2l2, and other antioxidant genes in HepG2 cells. (a) HepG2 cells were cotransfected for 8 h with mNfe2l1-luc reporter and the pRL-TK control, along with an expression construct for mouse α-pal^NRF1 or an empty pcDNA3.1, and then allowed for a 24 h recovery before the luciferase activity was measured (A1). Total cell lysates were also subjected to identification by Western blotting with distinct antibodies against α-Pal^NRF1 (A2) or V5 tag (A3). (b) HepG2 cells were cotransfected for 8 h with mNfe2l1-luc (B1) or hNfe2l1-luc (B2), plus pRL-TK, and then treated with 50 μmol/L tBHQ or the DMSO vehicle for 24 h, before being allowed for additional 24 h recovery. Subsequently, these samples were subjected to dual luciferase assays. The results are shown as () with significant increases ($, ). (c) HepG2 cells were cotransfected for 8 h with hNfe2l1-luc (C1) or ARE×6-luc (C2) together with pRL-TK, plus a human α-Pal^NRF1 expression construct or an empty pcDNA3.1 and then allowed for 24 h recovery from cotransfection, before the reporter activity was measured. The data are shown as () with significant decreases (). Total cell lysates were also subjected to characterization by Western blotting with distinct antibodies against α-Pal^NRF1 (C3) or V5 tag (C4). (d) Distinct protein levels of α-Pal^NRF1, TFAM, SOD1, Nfe2l1, Nfe2l2, HO-1, and GCLM were determined by Western blotting of HepG2 cells that had been transfected with α-Pal^NRF1 expression plasmid or an empty pcDNA3 vector. (e–l) Distinct changes in mRNA levels of α-Pal^NRF1 (e), TFAM (f), SOD1 (g), COX5a (h), Nfe2l1 (i), Nfe2l2 (j), HO-1 (k), and GCLM (l) were analyzed by real-time qPCR of HepG2 cells that had been transfected with 0, 25, 50, and 100 nM of siRNA against α-Pal^NRF1. The resulting data are shown as () with significant decreases (, ) or increases ($, ). (m) Such siRNA-transfected cell lysates were subjected to Western blotting analysis of distinct protein abundances as indicated. (n) A model is proposed to present disparate effects of α-pal^NRF1 overexpression or its knockdown on the nuclear-to-mitochondrial respiratory and antioxidant genes in HepG2 cells.

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