Fig. 6. HERV-K activation by TDP-43 and identification of binding sites on LTR. (A and B) Stem cell–derived neurons were transfected with either pcDNA control or TDP-43 expression construct. HERV-K activation by TDP-43 and identification of binding sites on LTR. (A and B) Stem cell–derived neurons were transfected with either pcDNA control or TDP-43 expression construct. (A) Forty-eight hours after transfection, cells were fixed with PFA and stained for HERV-K env protein. Scale bar, 50 μm. (B) Twenty-four hours after transfection, cells were collected for RNA extraction, and quantitative RT-PCR was used to measure HERV-K transcripts. (C and D) HERV-K plasmid was cotransfected with chloramphenicol acetyltransferase (CAT) (control), Tat, TDP-43, or Tat and TDP-43 in HeLa cells, and 24 hours after transfection, reverse transcriptase activity (HERV-K RT) was measured in culture supernatants by the product-enhanced reverse transcriptase (PERT) assay. (D) The levels of HERV-K transcripts were measured using RT-PCR and expressed as fold change compared to CAT control. (E) HERV-K LTR-MetLuc plasmid was cotransfected with CAT, Tat, TDP-43, or Tat and TDP-43, and luciferase activity was measured. RLU, relative light units. (F) Knockdown of endogenous TDP-43 with siRNA reduced HERV-K expression. (G) Putative TDP-43 binding sites in HERV-K LTR reported relative to the first base of the LTR. (H) Binding of TDP-43 to biotinylated oligonucleotides derived from the putative binding sites under low- or high-salt conditions. (I) Quantification of (H) indicating binding affinity. Values represent means ± SEM from three independent experiments. Significance was determined by unpaired Student’s t test. nt, nucleotide. Wenxue Li et al., Sci Transl Med 2015;7:307ra153 Published by AAAS