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

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 1 Depletion of p54nrb lowers the viability of tumor cells. P54nrb expression level in healthy tissue versus tumor tissue A from cervix carcinoma, B from colon carcinoma, and C from melanoma patients. Values were obtained from Oncomine database. To test significance Student´s t test was performed. ****p < 0.0001. Immunoblot of p54nrb-level in shRNA-control and shRNA-p54nrb#1 knockdown cells D from DLD-1 and E from SK-MEL. Counts of colonies of 3D soft agar tumor growth assay of shRNA-control and shRNA-p54nrb#1 cells F from DLD-1 and G from SK-MEL. 1000 cells/well were seeded and grown for 3 weeks and stained with 0.1% crystal-violet. Student´s t test was performed. *p < 0.05, n = 3 (G). Representative wells of 3D soft agar tumor growth assay of shRNA-control and shRNA-p54nrb#1 cells H from DLD-1 and I from SK-MEL cells. J Schematic representation of protein domains of p54nrb (also: NoNO): H and Q rich domain (HQ), RNA recognition motif (RRM), NonA/paraspeckle domain (NOPS), coiled-coil domain (COIL), and proline-rich region (P). Brackets indicate the region for RNA, DNA, and protein binding.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Expressing, Western Blot, shRNA, Control, Knockdown, Growth Assay, Staining, Protein Binding

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 2 Depletion of p54nrb leads to increased cell death susceptibility. A Immunoblot of p54nrb-level in shRNA-control and shRNA- p54nrb#3 knock down HeLa cells. B Flow cytometry of HeLa shRNA-control and shRNA-p54nrb#3 cells at 24 h after treatment with 10 µM etoposide (Eto) and 240 ng/ml human recombinant TRAIL (TRAIL). Cell viability was measured by detection and totaling of Annexin-V single positive and Annexin-V and propidium-iodide- double positive cells. The cells not falling to either of these categories were considered as viable cells. Significance was calculated with Student´s t test, *p < 0.05, n = 3. C Flow cytometry of HeLa shRNA-control and shRNA-p54nrb#3 cells at 24 h after treatment with 10 µg/ml Doxorubicin (DOX). Cell viability was measured by detection and totaling of Annexin-V single positive and Annexin-V and Sytox Blue- double-positive cells. The cells not falling to either of these categories were considered as viable cells. Significance was calculated with Student´s t test, **p < 0.01, n = 3.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, shRNA, Control, Knockdown, Flow Cytometry, Recombinant

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 3 Loss of p54nrb leads to an altered expressional pattern of carcinogenesis relevant genes. A Volcano plot diagram of LC/MS data derived from the analysis of HeLa shRNA-control versus shRNA-p54nrb cells. (n = 3). Arrowheads depict significantly (p < 0.01) upregulated (arrow up, red) and downregulated (arrow down, blue) proteins, and the total numbers of both categories are indicated on the diagram. B Up- and C downregulated proteins (p < 0.01, Log2 ratio either >0.5 or < −0.5) of HeLa-shRNA-p54nrb cells compared to shRNA control cells, which exert tumor regulatory and/or apoptosis regulatory properties. Differently colored oval diagrams represent various functional subcategories of these hits. D Immunoblot of HeLa shRNA-control and shRNA-p54nrb cells. Detection of p54nrb, gelsolin, cathepsin-Z, NQO1, and TPD52 levels. E Quantitative evaluation of immunoblots of p54nrb, gelsolin, cathepsin-Z, NQO1, TPD52, and CDKN2A from HeLa shRNA-control and shRNA-p54nrb#1 and #3 cells. The bands were normalized to the shRNA-control samples. Significance was calculated with one sample t test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 3.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Liquid Chromatography with Mass Spectroscopy, Derivative Assay, shRNA, Control, Functional Assay, Western Blot

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 4 P54nrb is cleaved upon apoptosis stimulation depending on the presence of caspase-2. A Immunoblot of p54nrb cleavage in HeLa cells at 24 h after treatment with DMSO or 20 µM Z-VDVAD-fmk (-1 h) and 250 nM staurosporine (STS). B Immunoblot of p54nrb cleavage and caspase expression in HeLa cells. HeLa cells were transfected either with 300 or 1000 ng/ml pcDNA3-caspase-2-Flag or pcDNA3-caspase-3- myc, and 24 h later were treated either with DMSO as vehicle control or 250 nM STS. The samples were harvested for Western blot at 24 h. C Immunoblot of p54nrb cleavage and caspase-2 in DLD-1 CRISPR-control or DLD-1 CRISPR–caspase-2 cells at 24 h after treatment with DMSO, 10 µM or 50 µM Eto. D Immunoblot of p54nrb cleavage and caspase-2 in HeLa shRNA-control or HeLa shRNA-caspase-2 cells at 24 h after treatment with DMSO, 250 nM STS, 1 µM STS or 10 µM Eto. E Immunoblot of p54nrb cleavage and caspase-2 in HeLa shRNA-control or HeLa shRNA-caspase-2 cells at 1, 3, 6, and 10 h after treatment with 1 µM STS. DMSO was used as vehicle control. F Immunoblot of p54nrb and caspase-2 of nuclear and cytoplasmic fraction of HeLa cells. Lamin-A was detected as nuclear marker.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, Expressing, Transfection, Control, CRISPR, shRNA, Marker

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 5 Caspase-2 directly interacts with p54nrb and cleaves p54nrb at D422. A Immunoblot of p54nrb caspase-2 and caspase-3 after endogenous immunoprecipitation (IP) of p54nrb from HeLa cells. B Immunoblot of p54nrb and caspase-2 after in vitro caspase cleavage assay with 0.2 µg or 2 µg recombinant p54nrb and 10 U human recombinant active caspase-2 after 3 h incubation at 37 °C. C Immunoblot of p54nrb cleavage and caspase-2 from HeLa cells at 48 h after ectopic expression of 1 µg/ml empty vector pcDNA3-Flag, pcDNA3-caspase-2-Flag, and pcDNA3-caspase-2-C303A-Flag (inactive). D Graphical illustration of p54nrb cleavage by activated caspase-2 via either overexpression, apoptotic induction, or recombinant caspase-2 protein. The active site of caspase-2 dimer is marked with yellow stars. Location of the putative cleavage sites in the p54nrb protein domains are marked with arrows, and its anticipated cleavage fragments are shown. E Immunoblot of p54nrb cleavage in HeLa shRNA-p54nrb#1 cells after ectopic expression of pcDNA3-Flag, pFlag-p54nrb, pFlag-p54nrb-D58N and pFlag- p54nrb-D422A (1 µg/ml plasmid) (+24 h) and 24 h treatment with 50 µM Eto. F Endogenous ribonucleoprotein immunoprecipitation of p54nrb in HeLa cells and subsequent isolation of coprecipitated DNA or RNA coupled with cDNA-synthesis. Validation of potential co- precipitate by PCR and subsequent agarose gel-electrophoretic detection. G In vitro p54nrb/DNA binding assay. 0.5 µg human recombinant p54nrb (Origene, Rockville, MD USA) and 100 ng plasmid encoding the sequence of gelsolin (Ch-gelsoln) [#37262] [Addgene]) were incubated at 37 °C for 18 h. Immunoprecipitation (IP) was performed by employing either p54nrb antibody or the same amount and species of IgG as control. Next, the potentially binding DNA was isolated as described in Methods (see ChIP). P54nrb was detected by western blot and the binding of specific DNA was confirmed by PCR, employing gelsolin-specific primers.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: Western Blot, Immunoprecipitation, In Vitro, Cleavage Assay, Recombinant, Incubation, Expressing, Plasmid Preparation, Over Expression, shRNA, Isolation, cDNA Synthesis, Biomarker Discovery, Agarose Gel Electrophoresis, DNA Binding Assay, Sequencing, Control, Binding Assay

Journal: Cell death & disease

Article Title: The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

doi: 10.1038/s41419-022-04829-2

Figure Lengend Snippet: Fig. 6 Cleavage by caspase-2 disrupts the gene regulatory function of p54nrb. A Schematic illustration of p54nrb’s gene regulatory function in the presence or absence of active caspase-2 and its subsequent effect on tumor cell survival.

Article Snippet: The following antibodies were employed: p54nrb/NONO (#A300-587A-1) (Bethyl, Montgomery, TX USA and caspase-2 (Cell Signaling Technology).

Techniques: