Reactivation of ERV reporters and ERVs upon siRNA-mediated KD of H3K9me3-binding proteins. (A) The percentage of enhanced green fluorescent protein-positive mESCs with reactivated ERV reporters was determined by flow cytometry (upper panel) on day 5 after the second transfection with siRNA against specified H3K9me3 readers. At least 10,000 cells were collected for each sample. Data are presented as means ± SD of three biological replicates. KD efficiency was determined by qRT-PCR at 30 hours after the second siRNA transfection (lower panel). Data are presented as means ± SD of three technical replicates. (B) Relative expression of ERVs at day 5 after the second KD with the indicated siRNA pool (upper panel), along with the efficiency of each KD, as determined by qRT-PCR at 30 hours after the second siRNA transfection (lower panel) is presented as means ± SD of three technical replicates. For each amplicon, expression was normalized to β-actin relative to scramble siRNA KD. (C) Western blot analysis of HP1 proteins in single- and triple-KD cells at day 5 after the second siRNA transfection is shown. H3 was used as a loading control.