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. 2011 Nov 8;21(12):1723–1739. doi: 10.1038/cr.2011.176

Figure 8.

Figure 8

UHRF1 but not UHRF2 interacts with DNMT1 in DG44-CHO cells and this interaction is S phase dependent. (A) The interactions between DNMT1 and UHRF1, and DNMT1 and UHRF2 were analyzed in DG44-CHO cells using CFP-Lac-UHRF1, CFP-lac-UHRF2 and Flag-DNMT1. Expression of control CFP-Lac, CFP-Lac-UHRF1 and CFP-Lac-UHRF2 all resulted in a bright foci in cells. However, recruitment of Flag-DNMT1 was observed only for CFP-Lac-UHRF1 cells in about 10% - 15% cells expressing both CFP-Lac-UHRF1 and Flag-DNMT1. (B) The interaction with DNMT1 was reciprocally analyzed using CFP-Lac-DNMT1 and Flag-UHRF1 and Flag-UHRF2 in DG44-CHO cells. The colocalization with CFP-Lac-DNMT1 was observed only for Flag-UHRF1 but not Flag-UHRF2. Note the colocalization was also observed on other regions enriched with CFP-Lac-UHRF1. This interaction is again observed only in 10% - 15% cells coexpressing both CFP-Lac-DNMT1 and Flag-UHRF1. (C) Aphidicolin treatment enriched DG44-CHO cells in S phase. DG44-CHO cells were treated with or without 1 mg/ml aphidicolin for 20 h. Aphidicolin was removed and cells were incubated in the presence of 10 μM EdU for 2 h and processed for EdU immunostaining (left panel) and merged figures with DAPI staining (right panel). (D) Aphidicolin treatment similarly increased the DG44-CHO cells with UHRF1 and DNMT1 colocalization.