Figure S1.
PC4 acts as an RBP that stabilizes CCND1 mRNA. Related to Fig. 1. (A) Pie chart showing the distribution of the PC4 RIP-seq reads in RNA classes. Data are generated from n = 2 biological replicates. (B) Volcano plot of the average difference of PC4-bound transcripts in NC and siPC4 groups determined by two-tailed Student’s t test from a linear model fit (x-axis). The y-axis indicates the P values. The genes of significantly downregulated (FC < −1, P < 0.05) are shown in red and upregulated (FC > 1, P < 0.05) genes are shown in blue. Vertical dashed lines indicate a cut-off of FC (1 or −1); horizontal dashed lines indicate a cut-off of P value (0.05). Data are generated from n = 2 biological replicates. (C) Heatmap representing the mRNA half-life of five indicated genes in Huh7 cells with control or overexpression of PC4, following treatment with ActD at different timepoints (hours). The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. (D) Western blot (left) and RIP-qPCR (right) showing the interaction between PC4 protein and CCND1 mRNA. Data were generated from n = 3 biological replicates. (E) RNA pull-down analysis showing the complex of PC4-CCND1 5′UTR in HepG2 cells. (F) Selected images of RNA-FISH. Huh7 cells expressing PC4-GFP were incubated with Cy5-labeled CCND1 5′UTR probe and stained with DAPI. PC4 protein is stained in green, CCND1 mRNA probe is stained in red, and DNA is stained with DAPI in blue. Scale bar = 40 μm; insert scale bar = 3.5 μm. (G) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in WDR74 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (H) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in LINC00869 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (I) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in CCND1 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (J) qPCR showing CCND2 mRNA expression in Huh7 and HepG2 cells with PC4 knockdown. Data in each group was normalized to that in NC. Data are generated from n = 3 biological replicates. (K) qPCR showing CCND3 mRNA expression in Huh7 and HepG2 cells with PC4 knockdown. Data in each group was normalized to that in NC. Data are generated from n = 3 biological replicates. (L) Western blot showing CCND2 and CCND3 protein expression in Huh7 and HepG2 cells with PC4 knockdown. The PC4 and β-actin blots are duplicated in Fig. 1 L. All graphed data are shown as means ± SD (one-way ANOVA test); error bars represent SD. ***P < 0.001. D–F and L are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

PC4 acts as an RBP that stabilizes CCND1 mRNA. Related to Fig. 1. (A) Pie chart showing the distribution of the PC4 RIP-seq reads in RNA classes. Data are generated from n = 2 biological replicates. (B) Volcano plot of the average difference of PC4-bound transcripts in NC and siPC4 groups determined by two-tailed Student’s t test from a linear model fit (x-axis). The y-axis indicates the P values. The genes of significantly downregulated (FC < −1, P < 0.05) are shown in red and upregulated (FC > 1, P < 0.05) genes are shown in blue. Vertical dashed lines indicate a cut-off of FC (1 or −1); horizontal dashed lines indicate a cut-off of P value (0.05). Data are generated from n = 2 biological replicates. (C) Heatmap representing the mRNA half-life of five indicated genes in Huh7 cells with control or overexpression of PC4, following treatment with ActD at different timepoints (hours). The color bar represents the level of mRNA expression. Data are generated from n = 3 biological replicates. (D) Western blot (left) and RIP-qPCR (right) showing the interaction between PC4 protein and CCND1 mRNA. Data were generated from n = 3 biological replicates. (E) RNA pull-down analysis showing the complex of PC4-CCND1 5′UTR in HepG2 cells. (F) Selected images of RNA-FISH. Huh7 cells expressing PC4-GFP were incubated with Cy5-labeled CCND1 5′UTR probe and stained with DAPI. PC4 protein is stained in green, CCND1 mRNA probe is stained in red, and DNA is stained with DAPI in blue. Scale bar = 40 μm; insert scale bar = 3.5 μm. (G) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in WDR74 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (H) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in LINC00869 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (I) IGV browser tracks showing PC4 and H3K27ac ChIP-seq reads in CCND1 DNA in Huh7 and HepG2 cells. PC4 ChIP-seq data are generated from n = 2 biological replicates. H3K27ac ChIP-seq data are generated from the dataset of GSM2360941 and GSM646355. (J) qPCR showing CCND2 mRNA expression in Huh7 and HepG2 cells with PC4 knockdown. Data in each group was normalized to that in NC. Data are generated from n = 3 biological replicates. (K) qPCR showing CCND3 mRNA expression in Huh7 and HepG2 cells with PC4 knockdown. Data in each group was normalized to that in NC. Data are generated from n = 3 biological replicates. (L) Western blot showing CCND2 and CCND3 protein expression in Huh7 and HepG2 cells with PC4 knockdown. The PC4 and β-actin blots are duplicated in Fig. 1 L. All graphed data are shown as means ± SD (one-way ANOVA test); error bars represent SD. ***P < 0.001. D–F and L are representative of three independent experiments. Source data are available for this figure: SourceData FS1.

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