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. 2023 Apr 26;14:1168589. doi: 10.3389/fimmu.2023.1168589

Figure 1.

Figure 1

TRIM5α H43Y efficiently blocks LINE-1 retrotransposition. (A) 293T or (B) 293T-shTRIM5α were transfected with LINE-1-GFP reporter plasmid or a retrotransposition-defective construct (JM111) together with empty vector or vector expressing the indicated single nucleotide polymorphism in TRIM5α. Expression of the HA-tagged TRIM5α constructs was confirmed by immunoblot using an HA-specific antibody. Five days posttransfection, GFP-positive cells were quantified by flow cytometry. The percentage of GFP-positive cells is presented as mean of triplicate transfections. Error bars represent SD. (C) 293T-shTRIM5α cells were transfected with LINE-1-GFP and the indicated TRIM5α variants. Five days posttransfection, genomic DNA was extracted and LINE-1 integration events were quantified by droplet digital PCR (ddPCR) using oligos targeting the spliced GFP reporter and normalized on the housekeeping gene RPP30 (copies per cell). Events in vector control cells are set to 100% (1.0). Results are shown as mean of quadruplicate transfections with error bars representing SD. One out of three independent experiments is shown. Statistical analysis were done using two-tailed, unpaired t-test. *P<0.1, **P<0.01, ***P<0.001, ****P<0.0001. R shRNA-resistant TRIM5α WT, H43Y, or R437C.