Figure 2.
Ubiquitination and phosphorylation of PC4 show periodic fluctuations and are associated with the stability of CCND1 mRNA. (A) Workflow for the identification of PC4 modifications during different cell cycle phases in Huh7 cells. Briefly, cells were treated with specific reagents to synchronize them in different cell cycle phases. After that, media was removed and cells were lysed. Then, the cell lysates were subjected to IP using the PC4 antibody. Subsequently, PC4 protein and its modifications were separated by SDS-PAGE before MS analysis. (B) Western blot showing the alternations of PC4 ubiquitination level at different cell cycle phases in PC4WT Huh7 cells. T/T, double-thymidine. HU, hydroxyurea. (C) Western blot showing the alternations of PC4 ubiquitination level at different cell cycle phases in PC4K68R Huh7 cells. (D) Western blot showing the changes of endogenous PC4 phosphorylation level at different cell cycle phases in Huh7 cells. (E) Western blot showing the presence of PC4 phosphorylation at asynchronous or synchronized M phase in Huh7 cells expressing Flag-tagged PC4 variant. (F) qPCR showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (G) qPCR showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (H) qPCR showing CCND1 mRNA expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (I) Western blot showing the indicated protein expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. The protein expressions are quantified and normalized, and the values are listed below each band. (J) qPCR showing CCND1 mRNA expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants at different cell cycle phases. Data are generated from n = 3 biological replicates. (K) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. B–E and I are representative of three independent experiments. Source data are available for this figure: SourceData F2.

Ubiquitination and phosphorylation of PC4 show periodic fluctuations and are associated with the stability of CCND1 mRNA. (A) Workflow for the identification of PC4 modifications during different cell cycle phases in Huh7 cells. Briefly, cells were treated with specific reagents to synchronize them in different cell cycle phases. After that, media was removed and cells were lysed. Then, the cell lysates were subjected to IP using the PC4 antibody. Subsequently, PC4 protein and its modifications were separated by SDS-PAGE before MS analysis. (B) Western blot showing the alternations of PC4 ubiquitination level at different cell cycle phases in PC4WT Huh7 cells. T/T, double-thymidine. HU, hydroxyurea. (C) Western blot showing the alternations of PC4 ubiquitination level at different cell cycle phases in PC4K68R Huh7 cells. (D) Western blot showing the changes of endogenous PC4 phosphorylation level at different cell cycle phases in Huh7 cells. (E) Western blot showing the presence of PC4 phosphorylation at asynchronous or synchronized M phase in Huh7 cells expressing Flag-tagged PC4 variant. (F) qPCR showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (G) qPCR showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (H) qPCR showing CCND1 mRNA expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. Data are generated from n = 3 biological replicates. (I) Western blot showing the indicated protein expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants. The protein expressions are quantified and normalized, and the values are listed below each band. (J) qPCR showing CCND1 mRNA expressions in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants at different cell cycle phases. Data are generated from n = 3 biological replicates. (K) Heatmap showing CCND1 mRNA stability upon ActD treatment in PC4-knockdown Huh7 cells stably expressing indicated Flag-tagged PC4 variants at different cell cycle phases. The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. All quantifications are shown as mean ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. B–E and I are representative of three independent experiments. Source data are available for this figure: SourceData F2.

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