Figure S2.
Ubiquitination and phosphorylation of PC4 show periodic fluctuations and are associated with the stability of CCND1 mRNA. Related to Fig. 2. (A) Western blot showing CCND1 and PC4 proteins expression at different cell cycle phases in Huh7 cells. (B) qPCR showing CCND1 and PC4 mRNA expression at different cell cycle phases in Huh7 cells. Data are generated from n = 3 biological replicates. (C)CCND1 mRNA stability upon ActD treatment in Huh7 cells with or without PC4 knockdown at different cell cycle phases. Data are generated from n = 3 biological replicates. (D) Liquid chromatography (LC)-MS/MS of PC4 ubiquitinated conjugation site. m/z, mass/charge ratio. (E) LC-MS/MS of PC4 phosphorylated conjugation site. m/z, mass/charge ratio. (F) Sequence alignment of PC4 residues aa 63–73 between different species using the Uniport alignment tool. (G) Schematic amino acid sequence of WT or Lys/Arg-mutant (K68R) PC4 protein. (H) Western blot showing different ubiquitination levels of PC4-Flag in Huh7 and HepG2 cells transfected with HA-tagged ubiquitin and indicated Flag-tagged PC4 variants. IB, immunoblot. (I) Western blot showing different ubiquitination levels of PC4-Flag in Huh7 cells transfected with indicated HA-tagged ubiquitin mutants and Flag-tagged PC4 variants. (J) Schematic amino acid sequence of WT or mutant (S17A/S19A/S17E) PC4 protein. (K) Western blot showing the phosphorylation level of PC4 in Huh7 cells expressing exogenous Flag-tagged PC4-WT, S17A, or S17E with endogenous PC4 knockdown. The p-Ser signal and Flag-tagged PC4 were examined with p-S17-PC4 and other indicated antibodies. (L) Western blot and statistical analysis showing PC4 protein stability upon cycloheximide (CHX) chase treatment at different timepoints in Huh7 cells expressing Flag-tagged PC4 variants. 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 < 0.0001. A, H, I, and K are representative of three independent experiments. Source data are available for this figure: SourceData FS2.

Ubiquitination and phosphorylation of PC4 show periodic fluctuations and are associated with the stability of CCND1 mRNA. Related to Fig. 2. (A) Western blot showing CCND1 and PC4 proteins expression at different cell cycle phases in Huh7 cells. (B) qPCR showing CCND1 and PC4 mRNA expression at different cell cycle phases in Huh7 cells. Data are generated from n = 3 biological replicates. (C)CCND1 mRNA stability upon ActD treatment in Huh7 cells with or without PC4 knockdown at different cell cycle phases. Data are generated from n = 3 biological replicates. (D) Liquid chromatography (LC)-MS/MS of PC4 ubiquitinated conjugation site. m/z, mass/charge ratio. (E) LC-MS/MS of PC4 phosphorylated conjugation site. m/z, mass/charge ratio. (F) Sequence alignment of PC4 residues aa 63–73 between different species using the Uniport alignment tool. (G) Schematic amino acid sequence of WT or Lys/Arg-mutant (K68R) PC4 protein. (H) Western blot showing different ubiquitination levels of PC4-Flag in Huh7 and HepG2 cells transfected with HA-tagged ubiquitin and indicated Flag-tagged PC4 variants. IB, immunoblot. (I) Western blot showing different ubiquitination levels of PC4-Flag in Huh7 cells transfected with indicated HA-tagged ubiquitin mutants and Flag-tagged PC4 variants. (J) Schematic amino acid sequence of WT or mutant (S17A/S19A/S17E) PC4 protein. (K) Western blot showing the phosphorylation level of PC4 in Huh7 cells expressing exogenous Flag-tagged PC4-WT, S17A, or S17E with endogenous PC4 knockdown. The p-Ser signal and Flag-tagged PC4 were examined with p-S17-PC4 and other indicated antibodies. (L) Western blot and statistical analysis showing PC4 protein stability upon cycloheximide (CHX) chase treatment at different timepoints in Huh7 cells expressing Flag-tagged PC4 variants. 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 < 0.0001. A, H, I, and K are representative of three independent experiments. Source data are available for this figure: SourceData FS2.

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