normal liver epithelial cells thle 3  (ATCC)

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

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: The effect of nobiletin (NOB) on the viability of THLE-2 and cholangiocyte cells after 24 h incubation. Data (mean ± SEM) from three independent experiments run in duplicate are presented.

Article Snippet: Human immortalized hepatocytes, THLE-2 (ATCC CRL-2706) cells, were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on the AST and ALT activity in THLE-2 ( A ) and cholangiocytes ( B ) cells. Data represent mean ± SEM from two independent experiments run in triplicate. Statistical significance was assessed using Dunnett’s test. The hashtag above denotes statistical significance relative to the treated INT+LPS-treated cells with # p < 0.05.

Article Snippet: Human immortalized hepatocytes, THLE-2 (ATCC CRL-2706) cells, were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on the JNK, p-JNK, NF-κB, p-NF-κB, STAT3, p-STAT3, in THLE-2 ( A ) and cholangiocytes ( B ) cells. Total protein and phosphorylated levels were quantified using fluorescence intensity (MFI) values obtained with the MAGPIX ® system. Data represent mean ± SEM from two independent experiments run in triplicate, and are expressed as fold change relative to untreated control cells (set to 100%). Statistical significance was assessed using Dunnett’s test. Asterisks above denotes statistical significance relative to the untreated control cells with * p < 0.05, ** p < 0.01, *** p < 0.001. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01.

Article Snippet: Human immortalized hepatocytes, THLE-2 (ATCC CRL-2706) cells, were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Control

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on PRKAA2 , SREBF2 , CYP7A1 , and ABCA1 mRNA levels after 24 h incubation. Data (mean ± SEM) represent relative mRNA expression in THLE-2 cells from two independent experiments run in triplicate, and are expressed as fold change relative to untreated control cells (set to 1). Statistical significance was assessed using Dunnett’s test. Asterisks above denotes statistical significance relative to the untreated control cells with ** p < 0.01, *** p < 0.001. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Human immortalized hepatocytes, THLE-2 (ATCC CRL-2706) cells, were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Expressing, Control

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on oxidative stress ( A ) and Nrf2 signaling pathways ( B ). ROS levels in THLE-2 cells after 24 h treatment with LPS, INT, INT+LPS, and INT+LPS+NOB (25 µM). Non-treated cells and DOXO (100 nM) served as negative and positive controls, respectively. ROS (+) and ROS (−) indicate cells with detectable or undetectable superoxide radicals, respectively. Representative histograms are shown, and data represent mean ± SEM from two independent experiments run in duplicate. Nrf2 subcellular distribution (cytosolic and nuclear fractions) and cytosolic SOD1 levels in THLE-2 cells after 24 h treatment. Representative blots are shown along with the corresponding Stain-Free™ total protein images used for total protein normalization (TPN). Lane order: (1) control, (2) LPS, (3) INT, (4) INT+LPS, (5) INT+LPS+NOB (10 µM), (6) INT+LPS+NOB (25 µM). Band intensities were quantified by densitometry and normalized to the lane-specific total protein signal obtained from Stain-Free imaging. Data are presented as mean ± SEM from two independent experiments, each performed in duplicate. Asterisks above denotes statistical significance relative to the untreated control cells with * p < 0.05, ** p < 0.01. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: Human immortalized hepatocytes, THLE-2 (ATCC CRL-2706) cells, were provided by the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Protein-Protein interactions, Staining, Control, Imaging

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: The effect of nobiletin (NOB) on the viability of THLE-2 and cholangiocyte cells after 24 h incubation. Data (mean ± SEM) from three independent experiments run in duplicate are presented.

Article Snippet: An important feature of this model is the implementation of the THLE-2 cell line (ATCC CRL-2706), which is derived from normal human hepatocytes immortalized with the SV40 large T antigen.

Techniques: Incubation

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on the AST and ALT activity in THLE-2 ( A ) and cholangiocytes ( B ) cells. Data represent mean ± SEM from two independent experiments run in triplicate. Statistical significance was assessed using Dunnett’s test. The hashtag above denotes statistical significance relative to the treated INT+LPS-treated cells with # p < 0.05.

Article Snippet: An important feature of this model is the implementation of the THLE-2 cell line (ATCC CRL-2706), which is derived from normal human hepatocytes immortalized with the SV40 large T antigen.

Techniques: Activity Assay

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on the JNK, p-JNK, NF-κB, p-NF-κB, STAT3, p-STAT3, in THLE-2 ( A ) and cholangiocytes ( B ) cells. Total protein and phosphorylated levels were quantified using fluorescence intensity (MFI) values obtained with the MAGPIX ® system. Data represent mean ± SEM from two independent experiments run in triplicate, and are expressed as fold change relative to untreated control cells (set to 100%). Statistical significance was assessed using Dunnett’s test. Asterisks above denotes statistical significance relative to the untreated control cells with * p < 0.05, ** p < 0.01, *** p < 0.001. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01.

Article Snippet: An important feature of this model is the implementation of the THLE-2 cell line (ATCC CRL-2706), which is derived from normal human hepatocytes immortalized with the SV40 large T antigen.

Techniques: Fluorescence, Control

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on PRKAA2 , SREBF2 , CYP7A1 , and ABCA1 mRNA levels after 24 h incubation. Data (mean ± SEM) represent relative mRNA expression in THLE-2 cells from two independent experiments run in triplicate, and are expressed as fold change relative to untreated control cells (set to 1). Statistical significance was assessed using Dunnett’s test. Asterisks above denotes statistical significance relative to the untreated control cells with ** p < 0.01, *** p < 0.001. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: An important feature of this model is the implementation of the THLE-2 cell line (ATCC CRL-2706), which is derived from normal human hepatocytes immortalized with the SV40 large T antigen.

Techniques: Incubation, Expressing, Control

Journal: Pharmaceutics

Article Title: Nobiletin Attenuates Inflammation and Modulates Lipid Metabolism in an In Vitro Model of Intestinal Failure-Associated Liver Disease

doi: 10.3390/pharmaceutics18010087

Figure Lengend Snippet: Effect of NOB, LPS, and INT on oxidative stress ( A ) and Nrf2 signaling pathways ( B ). ROS levels in THLE-2 cells after 24 h treatment with LPS, INT, INT+LPS, and INT+LPS+NOB (25 µM). Non-treated cells and DOXO (100 nM) served as negative and positive controls, respectively. ROS (+) and ROS (−) indicate cells with detectable or undetectable superoxide radicals, respectively. Representative histograms are shown, and data represent mean ± SEM from two independent experiments run in duplicate. Nrf2 subcellular distribution (cytosolic and nuclear fractions) and cytosolic SOD1 levels in THLE-2 cells after 24 h treatment. Representative blots are shown along with the corresponding Stain-Free™ total protein images used for total protein normalization (TPN). Lane order: (1) control, (2) LPS, (3) INT, (4) INT+LPS, (5) INT+LPS+NOB (10 µM), (6) INT+LPS+NOB (25 µM). Band intensities were quantified by densitometry and normalized to the lane-specific total protein signal obtained from Stain-Free imaging. Data are presented as mean ± SEM from two independent experiments, each performed in duplicate. Asterisks above denotes statistical significance relative to the untreated control cells with * p < 0.05, ** p < 0.01. Hashtags above denotes statistical significance relative to the INT+LPS-treated cells with # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: An important feature of this model is the implementation of the THLE-2 cell line (ATCC CRL-2706), which is derived from normal human hepatocytes immortalized with the SV40 large T antigen.

Techniques: Protein-Protein interactions, Staining, Control, Imaging