Journal: NAR Cancer

Article Title: Ribosome biogenesis is increased in hepatocellular carcinoma and represents a potential therapeutic target

doi: 10.1093/narcan/zcaf058

Figure Lengend Snippet: Nucleolar factors associated with FC/DFC are upregulated at both mRNA and protein level in HCC. Relative mRNA levels of ( A ) TCOF1 (Treacle), ( B ) UBF, ( C ) FBL (Fibrillarin), ( D ) NCL (nucleolin), and ( E ) NPM1 (nucleophosmin) measured by qPCR in HCC cells (PLC/PRF/5 and SNU-449) and control cells (THLE-3). GAPDH was used as the housekeeping gene, and fold change was determined relative to mRNA levels in THLE-3 cells. Individual points in the graph represent biological replicates ( n = 3). The graph depicts the mean ± SD, with statistical significance assessed by one-way ANOVA and indicated with numerical P -values. Protein levels of ( F ) Treacle, ( G ) UBF, ( H ) Fibrillarin, ( I ) nucleolin, and ( J ) nucleophosmin measured by WB in HCC cells (PLC/PRF/5 and SNU-449) and control cells (THLE-3). α-tubulin was used as a loading control (LC). The molecular weight in kDa is listed on the left side of the blots (left panel), and protein signals were determined in each cell line relative to the loading control (right panel). Individual points in the graphs represent biological replicates ( n = 3). The graph depicts the mean ± SD, with statistical significance assessed by one-way ANOVA and indicated by numerical P -values.

Article Snippet: Human osteosarcoma U2OS cells (#HTB-96), HCC cell lines PLC/PRF/5 (#CRL-8024), SNU-449 (#CRL-2234), and normal liver epithelial cells THLE-3 (#CRL-3583) were purchased from the American Type Culture Collection.

Techniques: Control, Molecular Weight

Journal: NAR Cancer

Article Title: Ribosome biogenesis is increased in hepatocellular carcinoma and represents a potential therapeutic target

doi: 10.1093/narcan/zcaf058

Figure Lengend Snippet: Nucleolar expression of FC/DFC-associated factors is upregulated in HCC. Representative images of ( A ) Treacle (green), ( B ) UBF (green), ( C ) Fibrillarin (green), and ( D ) nucleolin (green) and nucleophosmin (magenta), and Pol II (yellow) in THLE-3, PLC/PRF/5, and SNU-449 cells. ( E ) Total nucleolar Treacle intensity per nucleus. Graphs depict one representative replicate ( n = 3) with data points representing individual cells and the median indicated by the red line. A Kruskal–Wallis test was applied to assess differences between groups for all three biological replicates combined, and statistical significance is indicated by numerical P -values. ( F ) Total nucleolar UBF intensity per nucleus otherwise as in panel (E). ( G ) Total nucleolar Fibrillarin intensity per nucleus, otherwise as in panel (E). ( H ) Total nucleolar nucleolin intensity per nucleus otherwise as in panel (E). (I) Total nucleolar nucleophosmin intensity per nucleus otherwise as in panel (E).

Article Snippet: Human osteosarcoma U2OS cells (#HTB-96), HCC cell lines PLC/PRF/5 (#CRL-8024), SNU-449 (#CRL-2234), and normal liver epithelial cells THLE-3 (#CRL-3583) were purchased from the American Type Culture Collection.

Techniques: Expressing

Journal: NAR Cancer

Article Title: Ribosome biogenesis is increased in hepatocellular carcinoma and represents a potential therapeutic target

doi: 10.1093/narcan/zcaf058

Figure Lengend Snippet: rDNA transcription is increased in HCC. ( A ) Representative images of EU incorporation (green). THLE-3, PLC/PRF/5, and SNU-449 cells were stained with DAPI (blue) and an antibody against Pol II (yellow). ( B ) Total nucleolar EU intensity per nucleus as a readout for rDNA transcription in THLE-3, PLC/PRF/5, and SNU-449 cells. The graph depicts one representative biological replicate ( n = 3), with data points representing individual cells, and the median indicated by the red line. A Kruskal–Wallis test was applied to assess differences between groups for the replicates combined, and statistical significance is indicated by numerical P -values. ( C ) Nucleolar size measured through an inverse intensity-based mask of Pol II in THLE-3, PLC/PRF/5, and SNU-449 cells. Individual points in the graph represent biological replicates ( n = 3). The graph depicts the mean ± SD, and statistical significance assessed by one-way ANOVA is indicated with numerical P -values. ( D ) Number of nucleoli per nucleus in THLE-3, PLC/PRF/5, and SNU-449 cells measured through an inverse intensity-based mask of Pol II. Individual points in the graph represent biological replicates ( n = 3). The graph depicts the mean ± SD, and statistical significance assessed by one-way ANOVA is indicated with numerical P -values. ( E ) Total nucleolar area otherwise as in panel (C).

Article Snippet: Human osteosarcoma U2OS cells (#HTB-96), HCC cell lines PLC/PRF/5 (#CRL-8024), SNU-449 (#CRL-2234), and normal liver epithelial cells THLE-3 (#CRL-3583) were purchased from the American Type Culture Collection.

Techniques: Staining

Journal: NAR Cancer

Article Title: Ribosome biogenesis is increased in hepatocellular carcinoma and represents a potential therapeutic target

doi: 10.1093/narcan/zcaf058

Figure Lengend Snippet: Anti-cancer drugs targeting the nucleolus inhibit cell viability in HCC. Drug response curves with corresponding IC 50 values and 95% CIs. Drugs were applied for 72 h, after which cell viability was assessed in THLE-3, PLC/PRF/5, and SNU-449 cells. ( A ) Drug response curve for CX-5461 with obtained IC 50 values and 95% CI (left panel). The graph shows the obtained IC 50 values represented as the mean ± SD (right panel). Individual points in the graph represent biological replicates ( n = 3). Statistical significance was assessed by one-way ANOVA and are indicated with numerical P -values. ( B ) Drug response curve for BMH-21 otherwise as in panel (A). ( C ) Drug response curve for Oxaliplatin, otherwise as in panel (A). ( D ) Drug response curve for JP-1302 otherwise as in panel (A). ( E ) Drug response curve for Sorafenib otherwise as in panel (A).

Article Snippet: Human osteosarcoma U2OS cells (#HTB-96), HCC cell lines PLC/PRF/5 (#CRL-8024), SNU-449 (#CRL-2234), and normal liver epithelial cells THLE-3 (#CRL-3583) were purchased from the American Type Culture Collection.

Techniques:

Journal: NAR Cancer

Article Title: Ribosome biogenesis is increased in hepatocellular carcinoma and represents a potential therapeutic target

doi: 10.1093/narcan/zcaf058

Figure Lengend Snippet: Nucleolar-targeting compounds inhibit nucleolar activity and induce selective DNA damage. ( A ) Representative images of EU incorporation (green) in THLE-3, PLC/PRF/5, and SNU-449 cells 24 h after treatment with four anti-cancer drugs targeting the nucleolus: CX-5461, BMH-21, Oxaliplatin, and JP-1302, or targeted therapy Sorafenib. Cells were treated with DMSO as control and were stained with DAPI (blue) and Pol II (yellow). ( B ) Mean nucleolar EU intensity per nucleus as a readout for rDNA transcription in THLE-3, PLC/PRF/5, and SNU-449 cells 24 h after treatment with four anti-cancer drugs targeting the nucleolus: CX-5461, BMH-21, Oxaliplatin, and JP-1302, or targeted therapy Sorafenib. Cells were treated with DMSO as control. The graph depicts one representative biological replicate ( n = 3), with data points representing individual cells, and the median indicated by the red line. A Kruskal–Wallis test was applied to assess differences between groups for the replicates combined, and statistical significance is indicated by numerical P -values. ( C ) Representative images of nuclear 53BP1 and γH2AX foci accumulation in THLE-3, PLC/PRF/5, and SNU-449 cells 24 h after treatment with four anti-cancer drugs targeting the nucleolus: CX-5461, BMH-21, Oxaliplatin, and JP-1302, or targeted therapy Sorafenib. Cells were treated with DMSO as control, and stained with DAPI (blue), 53BP1 (yellow), and γH2AX (red). ( D ) Fold change of nuclear 53BP1 foci per cell relative to DMSO. Individual points in the graph represent biological replicates ( n = 3). The graph depicts the mean ± SD and statistical significance was assessed by one sample t test and indicated by numerical P -values. ( E ) Fold change of nuclear γH2AX foci per cell relative to DMSO otherwise as in panel (D).

Article Snippet: Human osteosarcoma U2OS cells (#HTB-96), HCC cell lines PLC/PRF/5 (#CRL-8024), SNU-449 (#CRL-2234), and normal liver epithelial cells THLE-3 (#CRL-3583) were purchased from the American Type Culture Collection.

Techniques: Activity Assay, Control, Staining