Journal: Oncotarget

Article Title: PLAGL1 gene function during hepatoma cells proliferation

doi: 10.18632/oncotarget.25996

Figure Lengend Snippet: Genomic profile of the chromosome regions where the PLAGL1 , p53 , PPARy and p21 genes map in four hepatoma cell-lines

Article Snippet: The human hepatoma cell lines HepG2, Huh7, PLC/PRF/5 and SkHep1 derived from patients with HCC were acquired from American Type Culture Collection.

Techniques:

Journal: Oncotarget

Article Title: PLAGL1 gene function during hepatoma cells proliferation

doi: 10.18632/oncotarget.25996

Figure Lengend Snippet: ( A ) Fibroblasts. ( B ) PLC/PRF/5. ( C ) SkHep1. ( D ) HepG2. ( E ) Huh7. The statistical comparisons were made between any given time point and T = 0 h for each cell line individually. Bars represent mean ± SD. Asterisks indicate that the difference is significant; * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The human hepatoma cell lines HepG2, Huh7, PLC/PRF/5 and SkHep1 derived from patients with HCC were acquired from American Type Culture Collection.

Techniques:

Journal: Oncotarget

Article Title: PLAGL1 gene function during hepatoma cells proliferation

doi: 10.18632/oncotarget.25996

Figure Lengend Snippet: The Western blot data corresponding to each time point was compared with that of T = 0 h. ( A ) Fibroblasts. ( B ) PLC/PRF/5. ( C ) SkHep1. ( D ) HepG2. ( E ) Huh7. A protein extract from fibroblasts (Fibr) was used as a signal control for the antibody p21. Representative blots are shown at the side of each graph. Actin was used as loading control. Bars represent mean ± SD. Asterisks indicate statistically significant differences; * p < 0.05, ** p < 0.001, *** p < 0.0001.

Article Snippet: The human hepatoma cell lines HepG2, Huh7, PLC/PRF/5 and SkHep1 derived from patients with HCC were acquired from American Type Culture Collection.

Techniques: Western Blot, Control

Journal: Oncotarget

Article Title: PLAGL1 gene function during hepatoma cells proliferation

doi: 10.18632/oncotarget.25996

Figure Lengend Snippet: Microscopic exam of the preparations revealed that PLAGL1 protein localized only in the cytoplasm of tumor cells, while p21 exhibited both nuclear and cytoplasmatic localization. Similar to Western blot analysis, this protein was not detected in Huh7 cells. ( A ) Fibroblasts; ( B ) PLC/PRF/5; ( C ) HepG2; ( D ) Huh7; ( E ) SkHep1; ( F ) Negative control. Magnification 1000× (A–E) and 400× (F).

Article Snippet: The human hepatoma cell lines HepG2, Huh7, PLC/PRF/5 and SkHep1 derived from patients with HCC were acquired from American Type Culture Collection.

Techniques: Western Blot, Negative Control

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: Gene expression of lipogenic enzymes and glycolysis-associated enzymes in three HCC cell lines. Gene expression analysis in three lipogenic enzymes through real-time PCR revealed that (A) FASN expression is significantly lower in SkHep1 cells than in Hep3B and HepG2 cells (Kruskal–Wallis test *** p < 0.001; n = 24 for each cell line), (B) ACSS2 expression is significantly higher in HepG2 cells than in Hep3B and SkHep1 cells (Kruskal–Wallis test *** p < 0.001; n = 12 for each cell line), and (C) ACLY expression (Kruskal–Wallis test * p = 0.016; n = 6 for each cell line) is significantly lower in SkHep1 cells than in Hep3B and HepG2 cells. (D) The expression of glycolysis-associated enzyme PDH in SkHep1 cells is significantly higher than that in Hep3B and HepG2 cells (Kruskal–Wallis test ** p = 0.002; n = 15 for each cell line). We summarized all the measurements as box-plots that label 25, 50, and 75% quantiles in the boxes with whiskers presenting at most 1.5 × interquartile range.

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Expressing

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: Dual-tracer uptake in three HCC cell lines in vitro and in vivo . In vitro , (A) 14 C-acetate uptake in HepG2 cells is significantly higher than that in SkHep1 and Hep3B cells (Kruskal–Wallis test *** p < 0.001; n = 7, 10, and 10 for HepG2, Hep3B, and SKHep1, respectively). (B) 18 F-FDG uptake in SkHep1 cells is significantly higher than that in HepG2 and Hep3B cells (Kruskal–Wallis test * p = 0.045; n = 6, 6, and 5 for HepG2, Hep3B, and SKHep1, respectively). In vivo , (C) 11 C-acetate micro-PET imaging of HCC xenografts in nude mice, represented by a color-based radiometric scale, revealed that (D) 11 C-acetate uptake assessed using the tumor-to-muscle ratio is significantly higher in HepG2 cells than in SkHep1 cells ( D ; permutation test *** p < 0.001; n = 6 and 5 for HepG2 and SkHep1, respectively); whereas (E) 18 F-FDG micro-PET imaging of HCC xenografts revealed that (F) 18 F-FDG uptake is significantly higher in SkHep1 cells than in HepG2 cells ( F ; permutation test *** p < 0.001; n = 5 for each cell line). The red arrows in dual-tracer micro-PET imaging in C,E indicate the location of the HCC xenografts. We summarized the measurements as box-plots that label 25, 50, and 75% quantiles in the boxes with whiskers presenting at most 1.5 × interquartile range.

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques: In Vitro, In Vivo, Micro-PET, Imaging

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: 14 C-acetate incorporated into cellular lipid fractions in three HCC cell lines. 14 C-acetate was incorporated into the cellular lipid fraction in the three HCC cell lines. In the extracted lipid fraction, 14 C-acetate uptake in HepG2 was significantly higher than that in Hep3B and SkHep1 (*** p < 0.001; permutation test; n = 6, 9, and 9 for HepG2, Hep3B, and SkHep1, respectively), indicating that 14 C-acetate was intensively accumulated in HepG2 cells and subsequently incorporated into cellular lipid fractions. 14 C-acetate uptake (β counter) in cells normalized by protein weight (CPM/mg protein). We summarized the measurements as box-plots that label 25, 50, and 75% quantiles in the boxes with whiskers presenting at most 1.5 × interquartile range.

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques:

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: Treatment response to orlistat treatment assessed using 14 C-acetate uptake and 14 C-acetate incorporated lipid fractions in three HCC cell lines. In HepG2 cells, (A) 14 C-acetate uptake and (B) 14 C-acetate incorporated lipid fractions were significantly inhibited by orlistat treatment (labeled by light gray) compared with mock treatment (labeled by dark gray) (permutation test *** p < 0.001 in both A,B ; n = 6 for each bar). However, orlistat-induced inhibition was not significant in Hep3B and SkHep1 (permutation test p = 0.236 and 0.630, respectively; n = 6 for each bar). 14 C-acetate uptake (β counter) in cells normalized by protein weight (CPM/mg protein). We summarized these measurements as bar plots that label the averaged values with error bars presenting the standard deviation (±1 SD) computed from six replicates for each treatment ( n = 6 for each bar).

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques: Labeling, Inhibition, Standard Deviation

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: Treatment response to orlistat assessed using cell cycle analysis with flow cytometry in three HCC cell lines. HepG2 cells in the S/G2/M cell cycle were obviously reduced after orlistat treatment compared with mock treatment, whereas no apparent change was observed in the cell cycle in Hep3B and SkHep1 cells. The proportion was determined by counting 2 × 10 5 cells for each treatment ( n = 2 × 10 5 ).

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques: Cell Cycle Assay, Flow Cytometry

Journal: Frontiers in Medicine

Article Title: Dual-tracer positron emission tomography/computed tomography as an imaging probe of de novo lipogenesis in preclinical models of hepatocellular carcinoma

doi: 10.3389/fmed.2022.1008200

Figure Lengend Snippet: Treatment response to orlistat in HCC xenografts assessed using tumor growth rates in nude mice. The tumor doubling time was significantly reduced in (A) orlistat-treated HepG2 xenograft mice compared with controls (* p = 0.015; n = 2 and 3 xenografts for control and orlistat treatment, respectively), whereas no significant difference was observed in (B) SkHep1 xenografts ( p = 0.561; n = 3 xenografts for each treatment). The open and solid circles are the averaged standardized tumor volumes for control and orlistat treatment, respectively. The averaged tumor volume as well as its error bar presenting the uncertainty (±1 SD) was calculated from the replicates observed at different times.

Article Snippet: To investigate the inhibitory effects of orlistat on HCC cells in vivo , we first established SkHep1 and HepG2 xenografts by inoculating 10 7 tumor cells subcutaneously into the shoulder of ten 7-week-old NOD/SCID mice (five in the experimental group and five in the control group; BioLASCO Taiwan).

Techniques: Control

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: Requirement of IGF-1R for p53 accumulation and activation in response to etoposide in MEFs. (A) Etoposide-mediated apoptosis is reduced in MEFs upon transfection with GFP-p53DD. R + and R − MEFs were transfected with either an empty vector or GFP-p53DD and treated with etoposide for 48 h. Apoptotic cell death was measured as described in . Values are mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01. The expression and activity of GFP-p53DD were determined using antibodies against GFP and p21. (B) Detection of p53 activation in MEFs. R + and R − MEFs were cotransfected with p53bs-luc (wild-type responsive elements) or p53ms-luc (mutant responsive elements) and a plasmid expressing renilla that served as an internal control. After etoposide treatments, luciferase activity was measured. Relative luciferase activity is expressed as light units normalized for renilla luciferase activity. Data shown are from one out of three independent experiments with comparable results (mean ± SD). (C) Induction of p53 and its downstream targets Mdm2 and p21 after etoposide treatment in R + and R − MEFs. R + and R − MEFs were treated with the indicated doses of etoposide for 12 h, and whole cell lysates were analyzed by Western blot analysis with the indicated antibodies. (D) AG1024 treatment impairs etoposide-induced p53 induction and activation in R + but not R − MEFs. R + and R − MEFs were pretreated with AG1024 before etoposide treatment. Whole cell lysates were harvested and subjected to Western blot analysis with the indicated antibodies. (E) Dose course of apoptosis induced by ionomycin in R + and R − MEFs. MEFs were subjected to different doses of ionomycin and collected after 36 h, and apoptosis was analyzed by flow cytometry. Values are mean ± SD from three independent experiments.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Activity Assay, Mutagenesis, Control, Luciferase, Western Blot, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: IGF-1R inhibition increases p53 protein stability. (A) Measurement of p53 protein stability in etoposide-treated MEFs. R + and R − MEFs were treated with etoposide for 24 h before exposure to CHX. Extracts prepared at the indicated times after the addition of CHX were analyzed by Western blot analysis (top). The stability of p53 protein was quantified by ImageQuant software (bottom). p53 band density was normalized to actin density, and then expressed relative to the t = 0 controls and plotted on a semilogarithmic scale by a linear regression program against the times of CHX treatments. Each decreased unit of log 2 (band density) is equivalent to one half life. (B) Measurement of the p53 protein stability in unstressed MEFs. R + and R − MEFs were treated with CHX for the indicated times. Quantitation of the stability of p53 protein was performed as described in A. Values are mean ± SD from three independent experiments. (C) Lack of IGF-1R leads to enhanced p53 protein stability. MEFs were pulse labeled with [ 35 S]methionine/cysteine and chased as described in Materials and methods. p53 protein was immunoprecipitated and resolved by SDS-PAGE (left) and the amount of 35 S was quantified by PhosphorImaging (right). (D) Conjugation of ubiquitin to p53 protein is reduced upon IGF-1R loss. R + and R − MEFs were harvested for immunoprecipitation. Equal amounts of immunoprecipitated p53 proteins were subjected to Western blot analysis with antibodies against ubiquitin.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Inhibition, Western Blot, Software, Quantitation Assay, Labeling, Immunoprecipitation, SDS Page, Conjugation Assay, Ubiquitin Proteomics

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: IGF-1R inhibition down-regulates Mdm2 expression at the translational level. (A) Measurement of Mdm2 expression in MEFs. Cell lysates were harvested and subjected to Western blot analysis with the indicated antibodies. (B) Down-regulation of Mdm2 by IGF-1R inhibition is independent of p53. p53 +/+ and p53 −/− HCT116 cells were incubated with or without AG1024. Expression of Mdm2 protein was determined by immunoblotting with the indicated antibodies. (C) Measurement of mdm2 mRNA levels upon AG1024 treatment. After AG1024 treatment, total cellular RNA was prepared and levels of mdm2 transcripts were revealed by semiquantitative RT-PCR. (D) IGF-1R inhibition reduces translation of mdm2 mRNA in a p53-independent manner. After AG1024 treatments, HCT116 p53 +/+ and p53 −/− cells were pulse labeled and newly synthesized. Mdm2 was immunoprecipitated from cells and analyzed by PhosphorImaging (top). Analysis of total cellular proteins by SDS-PAGE showed equal amounts of loading (bottom). (E) AG1024 treatment does not affect Mdm2 protein stability. HCT116 p53 +/+ cells were treated as described in C, and then exposed to CHX for the indicated times. Quantitative analysis of Mdm2 stability was performed as described in . Values are mean ± SD from three independent experiements. (F) HCT116 p53 +/+ cells were pulse labeled and chased for the indicated periods of time, followed by immunoprecipitation. The amount of 35 S was quantified by PhosphorImaging.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Inhibition, Expressing, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction, Labeling, Synthesized, Immunoprecipitation, SDS Page

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: The 5′ UTR of p53 and mdm2 transcript mediates IGF-1R–dependent regulation of p53 and Mdm2 translation. (A) AG1024 specifically reduces translational levels of p53. [ 35 S]methionine/cysteine pulse-labeled p27, c-fos, and p53 were immunoprecipitated from SK-hep1 cells treated with or without AG1024 and analyzed by PhosphorImaging (bottom). Expression levels of p27, c-fos, and p53 proteins were monitored by immunoblotting (top). Adjustment of brightness and contrast ( 35 S-p27 panel) was performed with Photoshop 8.0 software. (B) Chimeric UTR-luc constructs carrying the 5′ and/or 3′ UTR of p53, mdm2, and c-fos mRNA. Each construct is labeled with the corresponding number at the right side. (C) AG1024 inhibits p53 and Mdm2 translation at the level of translation initiation; a schematic presentation of the FLAG-p53, FLAG-Mdm2, and FLAG–c-fos constructs containing the corresponding UTRs, CrPV IRES, and EGFP (left). SK-hep1 cells were transfected with the indicated expression vectors. Cells were treated with or without AG1024 and analyzed for protein expression by immunoblotting with antibodies against FLAG and GFP. gfp mRNA levels were determined by semiquantitative RT-PCR. (D and E) 5′ UTRs of the p53 and mdm2 transcript impose IGF-1R signaling–mediated translational regulation. SK-hep1 cells were transiently cotransfected with the chimeric UTR-luc constructs and pRL–SV40–renilla as described in Material and methods. After AG1024 treatment, luciferase activity was measured. Relative luciferase activity is expressed as light units normalized for renilla luciferase activity. Data are presented as mean ± SD of three independent experiments performed in quadruplicate. Levels of luciferase transcripts were revealed by semiquantitative RT-PCR. The number at the bottom of the panel is representative of the corresponding chimeric construct shown in B.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Labeling, Immunoprecipitation, Expressing, Western Blot, Software, Construct, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Activity Assay

Journal: The Journal of Cell Biology

Article Title: A novel role for IGF-1R in p53-mediated apoptosis through translational modulation of the p53-Mdm2 feedback loop

doi: 10.1083/jcb.200703044

Figure Lengend Snippet: Reduced translational synthesis of p53 in R − MEFs. (A) Measurement of p53 mRNA levels in MEFs by Northern blot analysis. p53 mRNA levels were detected by Northern blot analysis in R + and R − MEFs. gapdh levels were shown as loading controls. (B) Reduced translation of p53 mRNA in R − MEFs. p53 protein was immunoprecipitated from R + and R − MEFs labeled with [ 35 S]methionine/cysteine and analyzed as described in (top, lanes 3 and 4). An SDS-PAGE gel confirmed equal loading of total cellular proteins.

Article Snippet: SK-hep1, HCT116 p53 +/+ , and p53 −/− cells (provided by B. Vogelstein, Johns Hopkins University, Baltimore, MD) were maintained in standard medium.

Techniques: Northern Blot, Immunoprecipitation, Labeling, SDS Page