Synchronized cells transfected with IMAGe-mutant CDKN1C show loss of oscillation of CDKN1C through the cell cycle. (A) Experimental outline for synchronization of HEK293T cells. Cells were synchronized with double thymidine block as on indicated. After the first block cells were transfected with a empty, wild-type CDKN1C, BWS-mutant CDKN1C (p.L42P) or IMAGE-mutant CDKN1C (p.K278E) and then synchronized again in early S phase with thymidine. After this process, a time was designated as “T0” and the cells were released and progressed through the S-phase (B) Upon release, cells were collected at the indicated times and CDKN1C was analyzed by immunoblotting. Beta-actin was used as a loading control. (C) Cell cycle analysis of double thymidine block synchronized cells transfected with empty plasmid, wild-type CDKN1C, BWS-mutant (p.L42P) or IMAGE-mutant (p.K278E) CDKN1C.