Fig. 5: FMDV VP4 impairs NME1-enhanced p53 transcriptional activity. | Cell Death & Disease

Fig. 5: FMDV VP4 impairs NME1-enhanced p53 transcriptional activity.

From: Foot-and-mouth disease virus induces lysosomal degradation of NME1 to impair p53-regulated interferon-inducible antiviral genes expression

Fig. 5

a HEK-293T cells cultured in 24-well plates were co-transfected with 0.2 μg Myc-vector or 0.2 μg Myc-NME1 and 0.1 μg Flag-vector or 0.1 μg Flag-p53 plasmids with Flag-vector (0.2 μg) or Flag-VP4 plasmids (0.2 μg), along with 0.1 μg p53 luciferase reporter plasmid. pRL-TK Renilla luciferase reporter plasmid (0.01 μg) was used in the reporter assay to normalize the transfection efficiency. The cells were collected at 24 hpt and luciferase activities were measured using the dual-specific luciferase assay kit. b HEK-293T cells cultured in 24-well plates were co-transfected with 0.2 μg Myc-vector or 0.2 μg Myc-NME1, and 0.2 μg Flag-vector or 0.2 μg Flag-VP4 plasmids, along with 0.1 μg p53 luciferase reporter plasmid and 0.01 μg pRL-TK plasmid. The transfected cells were incubated with DMSO or 5-FU (20 μg/mL) at 6 hpt, the luciferase activities were measured after 24 h incubation. c HEK-293T cells cultured in 24-well plates were co-transfected with 0.2 μg Myc-vector or 0.2 μg Myc-NME1, and increasing amounts of Flag-VP4 plasmids (0, 0.1, 0.2, or 0.4 μg), along with 0.1 μg p53 luciferase reporter and 0.01 μg pRL-TK plasmids. The transfected cells were incubated with 5-FU at 6 hpt, the luciferase activities were measured after 24 h incubation. Expression of Myc-NME1 and NME1 were detected by Western blotting using anti-NME1 antibody. Expression of Flag-VP4 was detected using anti-Flag antibody. d HEK-293T cells cultured in six-well plates were co-transfected with 2 μg Flag-vector or 2 μg Flag-VP4, and 2 μg Myc-NME1 plasimds. The transfected cells were treated with DMSO or 5-FU at 6 hpt for another 24 h. mRNA expression levels of ISG20, IRF9, RIG-I, and ISG15 were detected by qPCR. e Schematics of a series of Flag-tagged truncated VP4 constructs. f HEK-293T cells were co-transfected with Myc-NME1 and Flag-vector, Flag-tagged VP4 or VP4 mutant plasmids. Expression of My-NME1 was detected by Western blotting using anti-Myc antibody. Expression of Flag-VP4 and VP4 mutants was detected using anti-Flag antibody. Relative fold-change in abundance of Myc-NME1 protein in the transfectants was determined by densitometric analysis. g HEK-293T cells cultured in 24-well plates were co-transfected with 0.2 μg Myc-vector or 0.2 μg Myc-NME1, and Flag-VP4 or Flag-tagged VP4 mutants plasmids (0.2 μg), along with 0.1 μg p53 luciferase reporter and 0.01 μg pRL-TK plasmids. The transfected cells were incubated with 5-FU at 6 hpt, and luciferase activities were measured after 24 h incubation. All the experiments were repeated at least three times. h Amino acid alignment of different serotypes of FMDV VP4 coding sequences using LaserGene software (http://www.dnastar.com/). The GenBank accession numbers of the viral VP4 sequences were: Type O (AET43040.1), Type A (ADR66173.1), Type Asia 1 (ABF74751.2), Type C (ARO74648.1), Type SAT1 (ARO74655.1), Type SAT2 (ARO74650.1), and Type SAT3 (ALJ79273.1)

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