Figure 3

UBA52 regulates the cell cycle.

(A) Cell viability assays in UBA52-deficient cells. DLD-1 cells were transfected with a UBA52 siRNA. Then, cells were harvested for the cell viability assay at the indicated time. The fluorescent score was normalized to the level at 0 h. Data are representative of three independent experiments. **P < 0.01 (two-tailed Student’s t-test). (B) Cell-cycle analysis in UBA52-deficient cells. DLD-1 cells were transfected with UBA52 siRNA. Twenty-four hours later, cells were harvested for cell-cycle analysis. Data are representative of more than three independent experiments. **P < 0.01 (one-way ANOVA followed by Tukey’s test). (C) Myc-UBA52 (WT) regulates the cell cycle. DLD-1 cells were transfected with Myc-UBA52 (WT) #7R and UBA52 siRNA simultaneously. Thirty-six hours later, cells were harvested for cell-cycle analysis. Data are representative of more than three independent experiments. **P < 0.01 (one-way ANOVA followed by Tukey’s test). (D) Cell-cycle-related mRNA expression in UBA52-deficient cells. DLD-1 cells were transfected with a UBA52 siRNA. Twenty-four hours later, cells were harvested for quantitative real-time reverse-transcription PCR and normalized to ACTB levels. Data are representative of three independent experiments. (E) Cell cycle-related protein expression in UBA52-deficient cells. DLD-1 cells were transfected with a UBA52 siRNA. Twenty-four hours later, cells were harvested for immunoblotting. Data are representative of more than three independent experiments. (F) RPL40 co-localises with CDK6. DLD-1 cells were harvested for the in situ proximity ligation assay. Anti-UBA52 and anti-CDK6 antibodies were used. Data are representative of four independent experiments. **P < 0.01 (two-tailed Student’s t-test). (G) UBA52 interacts with CDK6. DLD-1 cells and HEK293T cells were transfected with FLAG-UBA52-GFP. Twenty hours later, cells were lysed and protein extracts were immunoprecipitated with GFP antibody and immunoblotted for the indicated proteins. Data are representative of more than three independent experiments in DLD-1 cells and HEK293T cells. (H) CHX chase experiment for the cyclin D protein. DLD-1 cells were treated with 100 μg/ml CHX and cell lysates were harvested for immunoblotting at the indicated time points. Data are representative of more than three independent experiments.