Figure 5.

Cytoplasm-localized hRFPL1 reduces cyclin B1 and Cdc2 accumulation to delay mitosis entry. (a, b) In FLAG-tagged hRFPL1-transduced HeLa cells, subcellular localization of hRFPL1 was determined by (a) immunocytochemistry using a pan-hRFPL1,2,3 antibody and (b) immunoblotting after subcellular compartment fractionation using a FLAG antibody. In (a), a similar subcellular localization was obtained by immunocytochemistry after endogenous induction of hRFPL1 expression by Pax6 overexpression (data not shown). In (b), the following abbreviations were used: cyto, cytosolic proteins; mb & org, proteins in membranes and organelles; nucl, nuclear proteins. Immunoblotting using EGFR, Golgi marker GM-130, eEF1A1 and Histone H3 antibodies was performed to control the fractionation process. (c, d) Cyclin B1 and Cdc2 accumulations (c) and number of cyclin B1-positive cells expressed as a percentage of the total number of cells (d) over the time were examined by immunoblotting and quantitative immunofluorescence, respectively, in eGFP- and hRFPL1-expressing HeLa cells following release from mimosine-induced G₁ phase block. In (d), ^***P<0.001 versus eGFP-expressing cells using two-way ANOVA followed by Tukey's post hoc test. (e, f) Cell-cycle parameters were assessed in eGFP- and hRFPL1-expressing HeLa cells using (e) EdU cumulative labeling and (f) determination of the percentage of labeled mitosis