ELG1 knockdown increases the level of monoubiquitinated PCNA in chromatin. A, HEK293T cells were transfected with various siRNAs (si-ELG1) targeting the coding region of the ELG1 mRNA or 3′-untranslated region (UTR) as indicated. Histone H4 protein was used as a loading control. PCNA-Ub, monoubiquitinated PCNA. B, nuclear extracts were prepared 48 h after transfection of HEK293T cells with siRNA targeting ELG1 or control siRNA and then subjected to immunoprecipitation with anti-PCNA antibody. Monoubiquitinated PCNA was identified with anti-ubiquitin (P4D1) antibody. An arrowhead indicates monoubiquitinated PCNA. C, the increase in PCNA monoubiquitination following ELG1 knockdown was not an off-target effect. HEK293T cells were cotransfected with the combination of siRNA targeting the 3′-UTR of ELG1 (#2 siRNA in A) and a FLAG-ELG1 expression vector as indicated. D, ELG1 knockdown does not affect cell cycle profile. HEK293T cells were transfected with siRNA targeting ELG1 or control siRNA twice with an interval of 24 h. Cells were harvested after 48 h from the second transfection, and their cell cycle profiles were determined by flow cytometry analysis. The x and y axes represent DNA content as measured by 7-amino-actinomycin D (7-AAD) staining and cells undergoing DNA replication as measured by bromodeoxyuridine incorporation, respectively. Each rectangle encompasses cells in G1, S, and G2/M phases of the cell cycle clockwise from the lower left. The numbers indicate the percentage of cells in the rectangle out of total cells. E, an increase in PCNA monoubiquitination was not observed following canonical RFC subunit (RFC1 or RFC4) knockdown. F, knockdown of other alternative RFC large subunits (CTF18 or RAD17) did not cause any significant change in PCNA monoubiquitination. The chromatin-bound fractions were prepared 72 h after transfection of HEK293T cells with the indicated siRNAs or a plasmid expressing FLAG-tagged ELG1 and then subjected to immunoblot analyses. The #3 siRNA in A that targets the coding region of ELG1 was used for ELG1 knockdown in B, E, and F.