Fig. 5

The interaction between Rif1 and PRC1.6 is mediated by Pcgf6. A The LacO-LacI induced colocalization experiments reveal that knockdown of different components of the PRC1.6 complex has no effect on the interaction between Rif1 and Pcgf6. GFP-Rif1 is shown in green, LacI-DsRed fused with Pcgf6 is shown in red, and DNA is stained with DAPI (blue). Bar: 10 μm. B RT-qPCR analysis of the indicated transcripts after the transfection of the corresponding shRNAs in the U2OS-LacO cell line. **** p < 0.0001 by t-test, Error bars represent SD, n = 3. C The relative enrichment of GFP-Rif1 in the indicated experimental groups. ns: not significant by t-test, Error bars represent SD, n = 10–23 cells per group. D The LacO-LacI induced colocalization experiments reveal that the interaction between Rif1 and RNF2 is weakened after the knockdown of Pcgf6. GFP-Rif1 is shown in green, LacI-DsRed fused with RNF2 is shown in red, and DNA is stained with DAPI (blue). Bar: 10 μm. E RT-qPCR analysis of Pcgf6 after the transfection of shRNAs targeting NT or Pcgf6 in the U2OS-LacO cell line. **** p < 0.0001 by t-test, Error bars represent SD, n = 3. F The relative enrichment of GFP-Rif1 in the indicated experimental groups. **** p < 0.0001 by t-test, Error bars represent SD, n = 29 in shNT group, n = 14 in shPcgf6 group. G Western blot showing the protein level of the indicated members of the PRC1.6 complex in the presence or absence of Rif1. H Coimmunoprecipitation of RNF2 and RYBP with endogenously tagged HA-Pcgf6 in the mESCs treated with shRNA targeting NT or Rif1. I Gel filtration of nuclear extracts from the Rif1 WT or Rif1 CKO mESCs. The corresponding elution volumes for each analyzed fraction are labeled. The arrow indicates the approximate elution volume of a 440 kDa protein complex