Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. Huh7, Hep3B and HepG2 liver cancer cells and immortalized human hepatocytes (THLE3) were incubated in the absence or in the presence of 5 ng/ml BMP9 in 0.1% FBS media and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.) are shown. B. THLE3 cells were incubated with different concentrations of BMP9 in 0.1% FBS media and counted after 4 days of treatment. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.) are shown. C. Proliferation curve of THLE3 cells incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS or in 10% FBS media. Data from one representative experiment (n = 3) out of 3 (mean ± S.D.) are shown. D. HepG2 cells were incubated with different concentrations of BMP9 in 0.1% FBS media and counted after 4 days of treatment. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). E. HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Data from 6 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t- test and data were compared to untreated samples, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. HepG2 cells were incubated for 1 hour with different concentrations of BMP9 in 0.1% FBS media. Western blots were performed with antibodies that recognized activated (phosphorylated) Smad1, 5 and 8 (P-Smad1,5,8) and Smad1 as loading control. A representative experiment of 2 is shown. B. HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Western blots were performed with antibodies that recognize Id1, P-Smad1,5,8 and total Smad1 (loading control). A representative experiment of 3 is shown. C. HepG2 stably expressing BRE-luciferase (HepG2-BRA) cells were plated and incubated with 0.1% FBS for 15 hours, and then treated with different concentrations of BMP9 for additional 15 hours. Luciferase activity was normalized to cell number. Data are shown as fold induction (relative to untreated cells) and are from one representative experiment (n = 4) out of 3 performed (mean ± S.D.). Statistical analysis was carried out using the paired t -test and data were compared to untreated samples, *** = P <0.001. D. HepG2 cells were incubated −/+ BMP9 (5 ng/ml) for different periods of time in 0.1% FBS media and Id1 levels were analyzed by qRT-PCR and normalized to 18S. Fold changes relative to untreated samples were determined (mean ± S.E.M, n = 3).

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Control, Stable Transfection, Expressing, Luciferase, Activity Assay, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A, B and C. HepG2 cells were incubated for 1 hour with A. dorsomorphin (1 µM, Dm), B. LDN193189 (100 nM) or C. ALK1ecd (16 fold molar excess, F.M.E.) and −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Western blots were performed with antibodies that recognize P-Smad1,5,8 and Smad1 as loading control. A representative experiment of 2 is shown in each case. D. HepG2 cells were incubated as in A and counted at day 4. Data from 2 independent experiments performed in triplicate (mean ± S.E.M.). E. HepG2 cells were incubated as in B and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). F. HepG2 cells were incubated as in C and counted at day 4. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). G. HepG2 cells were incubated without (C) or with dorsomorphin (Dm, 1 µM), LDN193189 (100 nM) or ALK1ecd (16 F.M.E) in 0.1% FBS media and counted at day 4. Data from at least 3 independent experiments performed in triplicate, displayed as percentage of C 0 samples (untreated cells, day = 0) (mean ± S.E.M). H. THLE3 cells were incubated with ALK1ecd (16 F.M.E) in 0.1% FBS media and counted at day 4. Data from 2 independent experiments performed in triplicate, displayed as percentage of C 0 (untreated cells, day = 0). Statistical analysis was carried out using paired t -test and data were compared to untreated samples, * = P <0.05, ** = P <0. 01, *** = P <0.001 or as indicated. n.s. = not significant.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Incubation, Western Blot, Control

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. Independent stable cell lines expressing non-silencing (N.S.) and two different shRNAs targeted against BMP9 were generated by retroviral infection of HepG2 cells. BMP9 mRNA levels were determined by quantitative RT-PCR and normalized to 18S. Data expressed relative to N.S. cells (assigned an arbitrary value of 1) from 3 different experiments (mean ± S.E.M). B. Non-silencing (NS), shBMP9#1 and #2 stable HepG2 cell lines were incubated in 0.5% FBS and −/+ BMP9 (5 ng/ml) and counted at day 6. Data from 6 independent experiments performed in triplicate, displayed as percentage of N.S. untreated cells (mean ± S.E.M). C. HepG2 cells were plated in soft agar and treated with BMP9 (5 ng/ml) or with dorsomorphin (Dm, 1 µM) for 3 weeks (added twice a week) and the colony number was counted. Data (n = 4, BMP9; n = 8, Dm) are displayed as percentage of control cells (mean ± S.E.M). D. Previously generated non-silencing (N.S.), shBMP9#1 and #2 stable HepG2 cell lines were plated in soft agar and counted after 3 weeks. Data from 4 experiments, displayed as percentage of N.S. cells (mean ± S.E.M). Statistical analysis was carried out using paired t -test and data were compared to untreated N.S. or control samples or as indicated, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: Stable Transfection, Expressing, Generated, Retroviral, Infection, Quantitative RT-PCR, Incubation, Control

Journal: PLoS ONE

Article Title: BMP9 Is a Proliferative and Survival Factor for Human Hepatocellular Carcinoma Cells

doi: 10.1371/journal.pone.0069535

Figure Lengend Snippet: A. DNA synthesis as determined by thymidine incorporation in HepG2 cells cultured for 24 hours in the absence or presence of BMP9 (5 ng/ml). Data are mean ± S.E.M. of 4 independent experiments and are displayed as percentage of untreated cells. B. HepG2 cells were incubated with or without BMP9 (5 ng/ml) in 0.1% FBS media or in the presence on 10% FBS media for 24 hours and then nuclear DNA content was analyzed by flow cytometry. Percentages of cells corresponding to the different cell cycle phases are shown. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t -test and data were compared to untreated samples (0.1% FBS), * = P <0.05, ** = P <0. 01, *** = P <0.001. C, D, E. HepG2 cells were treated as in B for 4 days. C–D. Cells were trypsinized and C. Nuclear DNA content was analyzed by flow cytometry. Percentages of hypodiploid (apoptotic) cells are shown. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). D. Cells were incubated with annexin V and PI. Subsequently, fluorescence intensity was measured in a FACScan flow cytometer and the percentage of annexin V positive/PI negative cells was calculated. Data from 3 independent experiments performed in triplicate (mean ± S.E.M.). E. Apoptotic nuclei were visualized and counted after PI staining under a fluorescence microscope. A minimum of 1000 nuclei was counted per condition. Data from 2 independent experiments performed in triplicate (mean ± S.E.M.). Statistical analysis was carried out using the paired t -test and data were compared to 10% FBS media treated samples or as indicated, * = P <0.05, ** = P <0. 01, *** = P <0.001.

Article Snippet: HepG2, Hep3B and Huh7 human HCC epithelial cells were obtained from the European Collection of Cell Cultures (ECACC), and non-tumoral human hepatocyte cell line THLE3 from the American Type Culture Collection (ATCC).

Techniques: DNA Synthesis, Cell Culture, Incubation, Flow Cytometry, Fluorescence, Staining, Microscopy