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. 2020 Nov 16;2020:5097109. doi: 10.1155/2020/5097109

Figure 5.

Figure 5

A negative effect of α-PalNRF1 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-palNRF1 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-PalNRF1 (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 mean ± SEM (n = 3 × 3) with significant increases ($, p < 0.01). (c) HepG2 cells were cotransfected for 8 h with hNfe2l1-luc (C1) or ARE×6-luc (C2) together with pRL-TK, plus a human α-PalNRF1 expression construct or an empty pcDNA3.1 and then allowed for 24 h recovery from cotransfection, before the reporter activity was measured. The data are shown as mean ± SEM (n = 3 × 3) with significant decreases (∗p < 0.01). Total cell lysates were also subjected to characterization by Western blotting with distinct antibodies against α-PalNRF1 (C3) or V5 tag (C4). (d) Distinct protein levels of α-PalNRF1, TFAM, SOD1, Nfe2l1, Nfe2l2, HO-1, and GCLM were determined by Western blotting of HepG2 cells that had been transfected with α-PalNRF1 expression plasmid or an empty pcDNA3 vector. (e–l) Distinct changes in mRNA levels of α-PalNRF1 (e), TFAM (f), SOD1 (g), COX5a (h), Nfe2l1 (i), Nfe2l2 (j), HO-1 (k), and GCLM (l) were analyzed by real-time qPCR of HepG2 cells that had been transfected with 0, 25, 50, and 100 nM of siRNA against α-PalNRF1. The resulting data are shown as mean ± SEM (n = 3 × 3) with significant decreases (∗p < 0.01, ∗∗p < 0.001) or increases ($, p < 0.01). (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-palNRF1 overexpression or its knockdown on the nuclear-to-mitochondrial respiratory and antioxidant genes in HepG2 cells.