Analysis of cytoplasmic granule formation by ZCCHC3 and L1 ORF1p.

(A) Endogenous ORF1p forms granules in untreated, (B) and larger granules in arsenite-stressed 2102Ep cells. However, endogenous ZCCHC3 colocalizes with LINE-1 ORF1 granules in treated 2102Ep cells only (B). (C) However, unlike in untreated 2102Ep cells, endogenous LINE-1 ORF1p does not enter cytoplasmic granules in untreated U2OS-WT cells, (D) but only after Na-arsenite stress. (E) Bar graphs summarize the data of micrograph experiments (F-I). Data were generated by counting at least 200 cells for each transfection. n = number of experimental replicates. GFP-tagged L1 ORF1p expressed from the construct ORF1-EGFP-L1-RP is able to form cytoplasmic granules in both unstressed or stressed (F,G) U2OS-WT or (H,I) U2OS-DKO cells. NT: no treatment. Cell nuclei were stained with Hoechst 33342 (right-most panels). Size bars are 10 μm.