Figure 5.
PC4 promotes G1–S transition and cell proliferation partially through a CCND1-dependent manner. (A) Asynchronized cell cycle analysis of Huh7 cells stably expressing control or PC4-KO. The number on the upper right corner represents the percentage of cells in different cell cycle phases. Data are generated from n = 3 biological replicates. (B) Synchronized cell cycle analysis of Huh7 cells with control or PC4 knockdown. Cells were treated with double-thymidine block, followed by release for indicated period of time. Data were generated from n = 3 biological replicates. (C) Western blot showing PC4 expression level in Huh7-FUCCI cells transfected with indicated siRNAs or plasmids. (D) Illustration of FUCCI-expressing Huh7 cells at each cell cycle phase. Cells at G1 display yellow fluorescence from mKO2, while cells at non-G1 phases (S, G2, and M) display green fluorescence from Clover. (E) The time length of the G1 phase in F. n = 30 cells in each group. Open circle represents an individual cell. (F) Representative frames of cell cycle tracking in FUCCI-expressing Huh7 cells transfected with indicated siRNAs or plasmids via time-lapse imaging. Scale bar = 50 μm. (G) Cell viability analysis by CCK8 assay in Huh7 and HepG2 cells with PC4 stable knockdown. Data are generated from n = 5 biological replicates. (H) Photographs of dissected tumors in the nude mice subcutaneously injected with Huh7 cell stably expressing NC, shPC4-1, shPC4-2, or shPC4+PC4 (the sequence for PC4 induction is optimized and resistant to shRNA). Data are generated from n = 5 biological replicates. (I) Tumor volume in the nude mice subcutaneously injected with Huh7 cell stably expressing NC, shPC4-1, shPC4-2, or shPC4+PC4. Data are generated from n = 5. (J) Cell viability analysis by CCK8 assay in Huh7 cells with NC, siPC4, siCCND1, or siPC4+siCCND1. Data are generated from n = 5 biological replicates. (K) The lengths of time from G1 to S phase in FUCCI-expressing Huh7 cells treated with NC, siPC4, siCCND1, or siPC4+siCCND1. Data are generated from n = 30 for each group. (L) Cell viability analysis by CCK8 assay in Huh7 and HepG2 cells with empty vector (NC), PC4 stable knockdown (shPC4), or cyclin D1 complemented PC4 knockdown (shPC4+CCND1). Data are generated from n = 5 biological replicates. (M) The lengths of time from G1 to S phase in FUCCI-expressing Huh7 cells treated with NC, shPC4, or shPC4+CCND1. Data are generated from n = 30 for each group. All quantifications were shown as means ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C is representative of three independent experiments. Source data are available for this figure: SourceData F5.

PC4 promotes G1–S transition and cell proliferation partially through a CCND1-dependent manner. (A) Asynchronized cell cycle analysis of Huh7 cells stably expressing control or PC4-KO. The number on the upper right corner represents the percentage of cells in different cell cycle phases. Data are generated from n = 3 biological replicates. (B) Synchronized cell cycle analysis of Huh7 cells with control or PC4 knockdown. Cells were treated with double-thymidine block, followed by release for indicated period of time. Data were generated from n = 3 biological replicates. (C) Western blot showing PC4 expression level in Huh7-FUCCI cells transfected with indicated siRNAs or plasmids. (D) Illustration of FUCCI-expressing Huh7 cells at each cell cycle phase. Cells at G1 display yellow fluorescence from mKO2, while cells at non-G1 phases (S, G2, and M) display green fluorescence from Clover. (E) The time length of the G1 phase in F. n = 30 cells in each group. Open circle represents an individual cell. (F) Representative frames of cell cycle tracking in FUCCI-expressing Huh7 cells transfected with indicated siRNAs or plasmids via time-lapse imaging. Scale bar = 50 μm. (G) Cell viability analysis by CCK8 assay in Huh7 and HepG2 cells with PC4 stable knockdown. Data are generated from n = 5 biological replicates. (H) Photographs of dissected tumors in the nude mice subcutaneously injected with Huh7 cell stably expressing NC, shPC4-1, shPC4-2, or shPC4+PC4 (the sequence for PC4 induction is optimized and resistant to shRNA). Data are generated from n = 5 biological replicates. (I) Tumor volume in the nude mice subcutaneously injected with Huh7 cell stably expressing NC, shPC4-1, shPC4-2, or shPC4+PC4. Data are generated from n = 5. (J) Cell viability analysis by CCK8 assay in Huh7 cells with NC, siPC4, siCCND1, or siPC4+siCCND1. Data are generated from n = 5 biological replicates. (K) The lengths of time from G1 to S phase in FUCCI-expressing Huh7 cells treated with NC, siPC4, siCCND1, or siPC4+siCCND1. Data are generated from n = 30 for each group. (L) Cell viability analysis by CCK8 assay in Huh7 and HepG2 cells with empty vector (NC), PC4 stable knockdown (shPC4), or cyclin D1 complemented PC4 knockdown (shPC4+CCND1). Data are generated from n = 5 biological replicates. (M) The lengths of time from G1 to S phase in FUCCI-expressing Huh7 cells treated with NC, shPC4, or shPC4+CCND1. Data are generated from n = 30 for each group. All quantifications were shown as means ± SD (one-way ANOVA test); error bars represent SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C is representative of three independent experiments. Source data are available for this figure: SourceData F5.

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