The HUSH complex is critical for endogenous retrotransposon repression in naïve pluripotent cells. (A) Endogenous retrotransposon expression was measured by qRT-PCR following shRNA-depletion of epigenetic modifiers in mESCs. (B) Naïve cells express higher levels (++) of ZFP42 (REX1) and NANOG (left), the latter shown by Western blot in two mESC strains (right). Predicted band sizes: NANOG, 34 kDa; PCNA, 29 kDa. (C) Endogenous retrotransposon expression following depletion of epigenetic modifiers. One representative experiment of three is shown. Atrx was not examined in the third experiment, excluding it from statistical analyses. Two-tailed paired t-tests were done for 2i + LIF samples. (D) Western blot for IAP GAG p73 using a rabbit IAP GAG antibody or PCNA as control in 2i + LIF J1 ESCs. The antibody detects p73 as well as GAG-POL and GAG cleavage products, including p41, representing partially processed GAG. Samples were re-run on a second gel and reblotted for L1 ORF1 protein (40 kDa) and reprobed for PCNA. (E) J1 ESCS grown in serum versus 2i conditions were blotted for epigenetic factors or PCNA as a normalizer. Predicted band sizes: SETDB1, 143 kDa; MPHOSPH8, 97 kDa; ATRX, 280 kDa; FAM208A, 200 kDa; KAP1, 100 kDa.
