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. 2017 Mar 15;45(8):4619–4631. doi: 10.1093/nar/gkx178

Figure 3.

Figure 3.

TREX1 suppresses L1 activity and L1-induced genome nicking by reducing L1 ORF1p levels. (A) Comet assay results concerning TREX1 suppression of L1-induced genome damage. HeLa cells were transfected with L1-1FH (2 μg), along with 500 ng control vector or the vector expressing TREX1 or one of its mutants on a 12-well plate. The cells were then subjected to the comet assay and fluorescent imaging at 96 h post-transfection. The data are representative of three independent experiments and three random areas are shown for each sample from the same experiment. (B) The tail moment of the comets in A was analyzed for 100 cells for each sample using Comet Assay IV software. (C) Western blotting results indicating corresponding levels of TREX1 in each sample shown in A. (D) Western blotting results showing that TREX1 does not reduce the protein levels of ORF2p. TREX1 (500 ng) or SAMHD1 (500 ng) expression plasmids were co-transfected with L1-2FH (2 μg) into HEK293T cells seeded on a 12-well plate. At 48 h post transfection, the cells were harvested for western blotting. SAMHD1 was previously reported to reduce ORF2p and is shown as a positive control. (E) Western blotting results showing that TREX1 potently reduces protein levels of ORF1p. TREX1 (500 ng) or SAMHD1 (500 ng) or MOV10 (500 ng) expression plasmids were co-transfected with L1-1FH (300 ng) into HEK293T cells seeded on a 12-well plate. At 48 h post -transfection, the cells were harvested for western blotting. SAMHD1 was introduced as a negative control, and MOV10 as a positive control (30,59). (F) Western blotting results showing that exonuclease-deactivated TREX1 mutants maintain the ability to reduce ORF1p levels. Five hundred nanogram TREX1 WT or DNase mutant expression plasmids were co-transfected with L1-1FH (300 ng) into HEK293T cells seeded on a 12-well plate. At 48 h post transfection, the cells were harvested for western blotting. (G) Western blotting results showing that AGS-associated mutations compromise TREX1's ability to deplete ORF1p. Vectors expressing TREX1 or its AGS mutants in a dose manner (50, 150 and 450 ng) were co-transfected with L1-1FH (300 ng) into HEK293T cells seeded on a 12-well plate. At 48 h post transfection, the cells were harvested for western blotting. (H) L1 assay results indicating that the suppressive effects of TREX1 and SAMHD1 against L1 replication are additive. Vectors expressing TREX1 (500 ng) or SAMHD1 (500 ng) or both plasmids were co-transfected with 2 μg L1-RP into HEK293T cells seeded on a 12-well plate to examine possible potency against L1 retrotransposition. The western blotting results show the expressed levels of TREX1 and SAMHD1. All the data shown in this figure are representative of at least three independent experiments. Three random areas are shown for each sample from the same experiment in A. The error bars shown in B indicate the standard error of the mean (SEM) of three independent experiments, and those in H indicate the SD of three replicates within one experiment.