Fig. 1

Cytoplasmic localization of L1 ORF1 protein. Stress granule marker proteins TIA1 (a, b) and eIF3η (c, d) show minimal colocalization with endogenous ORF1p in cytoplasmic granules (shown by arrows) of unstressed human embryonal carcinoma 2102Ep cells (a, c) but colocalize in large stress granules of cells treated with Na-arsenite (0.5 mM for 1 hour) (b, d). Cell nuclei are stained with Hoechst. Size bars are 10 μm. e ORF1p cytoplasmic granules retain integrity during cellular mitosis. Patient sera-derived α-ANA-N marks nucleoli [60]. Endogenous ORF1p is mostly excluded from metaphase chromatin plates (arrow), as shown by Hoechst staining (see arrow). f Ectopically expressed EGFP-tagged human ORF1p induces prominent cytoplasmic granules, but (g) deletion of its Q-N-rich region abolishes granule formation. h The ORF1p R159H point mutation reduces cytoplasmic granule formation by 50%. Approximately 500 cells were examined. i The R159H mutation abolishes cell culture LINE-1 retrotansposition. pc6-RPS-EGFP-ΔCMV wild-type or R159H mutant retrotransposition reporter constructs were transfected in HEK 293T cells and 5 days later the percentages of EGFP-positive cells were determined by flow cytometry. The construct 99-PUR-JM111-EGFP served as a negative control for retrotransposition [84]. Each construct was tested in quadruplicate wells with results for one biological replicate shown