Functional interaction between CRL4s^MMS22L and ESCO2.

(A) Over-expression of ESCO1, ESCO2 or PCNA partially suppresses a cohesion defect in DDB1 knockdown cells, while over-expression of ESCO2-LG mutant does not. Immunoblots of DDB1, ESCO1, ESCO2, PCNA and Tubulin were shown below each column. The statistical significance was calculated via student’s t-test, ** PS4A Fig for the corresponding CEN cohesion defect results. (B) ESCO1, ESCO2 or PCNA over-expression partially compensates for a SCC defect caused by MMS22L knockdown, while over-expression of ESCO2-LG mutant does not. Immunoblots of MMS22L, ESCO1, ESCO2, PCNA and Tubulin are shown below each column. The statistical significance was calculated via student’s t-test, ** PS4B Fig for the corresponding CEN cohesion defect results. (C) Over-expression of CUL4A, CUL4B, DDB1, or MMS22L partially suppresses the SCC defect caused by ESCO2 knockdown, while over-expression of an E3-interaction defective mutant, or ESCO2-LG (L415DG417D) mutant has no effect. ESCO2 was knocked down by siRNA in 293T cells, then Flag-tagged ESCO1, ESCO2, ESCO2-LG, CUL4A, CUL4B, DDB1, or MMS22L were overexpressed after siRNA delivery for 6 h. The percentage of cells in cohesion was determined as in Fig 1. Immunoblots of ESCO2, Flag and Tubulin from each RNAi experiment are shown below the corresponding column. The statistical significance was calculated via student’s t-test, *** PS4C Fig for the corresponding CEN cohesion defect results. See S4D Fig for the results of PCNA and PCNA-A252V alleles. (D) Over-expression of CUL4A, CUL4B, DDB1, MMS22L or PCNA suppresses the M phase arrest caused by ESCO2 knockdown. The portion of M-phase cells among total cells was counted after Hoechst 33342 staining. The statistical significance was calculated via student’s t-test, *** P<0.001, n: the total cell number counted.