Fig. 5

Increasing expression of some ALS-associated proteins alters retrotransposition in cell culture assays. n=number of biological replicates. a L1-RP is retrotransposition-competent in human SH-SY5Y neuroblastoma and mouse NSC-34 motor neuron-like cells. Cells in 6-well plates were infected with A/RT-pgk-L1RP-EGFP (Ad-L1) L1-reporter adenovirus [97] at about 8 × 10¹² viral particles/ml. Flow cytometry analysis was performed at 9 days post-infection. b 99-PUR-RPS-EGFP was co-transfected in HEK 293T cells with empty vector (pcDNA3) or test constructs expressing tagged ALS-related proteins. Five days later, percentages of EGFP-positive cells were determined by flow cytometry. Each plasmid pair was transfected in four replicate wells, with at least 3 replicate experiments performed for each construct. Results are normalized to pcDNA3 vector control (lighter bar). Statistical significances compared with vector control were calculated by Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). All test proteins were expressed as confirmed by Western blotting of whole cell lysates using α-DYKDDDDK (FLAG)-tag, α-Myc-tag, or α-V5-tag antibodies as indicated (top). Four-fold less V5-SQSTM1- and three-fold more V5-TBK1-transfected lysates were loaded on the gel. Full-length TBK1 expressed poorly, most of the protein existing as a high molecular weight smear. c FLAG-tagged TDP-43 co-immunoprecipitates T7-tagged L1 ORF1p complexes from HEK 293T cell lysates after α-FLAG-M2 affinity gel purification. Interaction is lost following treatment of lysates with RNase. d Expression of V5- or Myc-tagged TDP-43 strongly inhibits mouse IAP element retrotransposition in HeLa-JVM cell culture. Cells were treated with neomycin (G418) to select for retrotransposition events. Colony counts are not normalized. On the right are representative T₇₅ flask images with Giemsa-stained IAP retrotransposition-positive colonies in the absence or presence of TDP-43. The apparent diminished effect on retrotransposition of Myc-TDP-43 compared with TDP-43-V5-WT is likely because its plasmid backbone does not replicate and is diluted out of cells during the course of antibiotic selection which spans a couple of weeks