Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Comparison of Nanog, COMMD7, COMMD1, NF-κB, and HNF4α Expression between HCC and Adjacent Tissues (A) Expression of Nanog, COMMD7, COMMD1, and HNF4α in hepatocellular carcinoma (HCC) (cancer tissues [CTs]), compared with matched adjacent tissues (ATs). (B) Correlation analysis revealed a significant negative correlation between COMMD7 and COMMD1, and between COMM7 and HNF4α. (C) Western blot assays showed upregulation of Nanog, COMMD7, and NF-κB, and downregulation of COMMD1 and HNF4α in CT, compared with AT samples. (D) COMMD7 and COMMD1 expression in CT and AT samples using immunohistochemistry. Representative images from independent experiments are shown (×200 original magnification; scale bars, 500 μm.). ***p < 0.001 versus AT.

Article Snippet: For immunofluorescence, cells were fixed in 4% paraformaldehyde and then washed with PBS for 5 min. Next, cells were incubated with primary antibody against COMMD7, COMMD1, p65, or HNF4α at 4°C overnight, followed by incubation with FITC-labeled goat against secondary antibody (Beyotime Institute of Biotechnology).

Techniques: Comparison, Expressing, Western Blot, Immunohistochemistry

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Expression of COMMD7, COMMD1, NF-κB, and HNF4α in Huh7, HL-7702, and Nanog + HCSCs (A) Expression of Nanog, COMMD7, COMMD1, and HNF4α in Huh7, HL-7702, and Nanog + HCSCs. (B) Protein level of Nanog, COMMD7, COMMD1, NF-κB p65, and HNF4α. (C) Co-localization of COMMD7 and COMMD1. (D) Co-localization of COMMD7 and NF-κB p65. Scale bars, 50 μm. **p < 0.01; ***p < 0.001.

Article Snippet: For immunofluorescence, cells were fixed in 4% paraformaldehyde and then washed with PBS for 5 min. Next, cells were incubated with primary antibody against COMMD7, COMMD1, p65, or HNF4α at 4°C overnight, followed by incubation with FITC-labeled goat against secondary antibody (Beyotime Institute of Biotechnology).

Techniques: Expressing

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: The Effect of Stable Transfection of COMMD7 shRNA on Cell Proliferation, Apoptosis, Migration, and Invasion in Nanog + HCSCs (A and B) The expression of COMMD7 (A) and COMMD1 (B) mRNA was measured in Nanog + HCSCs after COMMD7 knockdown using real-time qPCR analysis. (C) Western blot assays analysis of Nanog, COMMD1, NF-κB, and HNF4α. (D) Immunofluorescence images of COMMD7 and COMMD1. (E) CCK-8 assay was performed to evaluate proliferative ability. (F and G) Hoechst 33258 staining (F) and flow cytometry assay (G) were performed to quantify the apoptotic rate. (H) Cell migration and invasion ability determined by wound healing and transwell invasion assay. (I) The subcutaneous xenograft murine model and representative images of tumors formed in the mice implanted with sh-COMMD7 or blank lentivirus-transfected Nanog + HCSCs. (J) Changes in the tumor volume every 2 days from day 10 until day 26 after the cells were implanted subcutaneously (n = 10). (K) Expression analysis of COMMD7, COMMD1, NF-κB, and HNF4α in tumor tissues collected from mice. Scale bars, 50 μm. *p < 0.05; ***p < 0.001 versus vector.

Article Snippet: For immunofluorescence, cells were fixed in 4% paraformaldehyde and then washed with PBS for 5 min. Next, cells were incubated with primary antibody against COMMD7, COMMD1, p65, or HNF4α at 4°C overnight, followed by incubation with FITC-labeled goat against secondary antibody (Beyotime Institute of Biotechnology).

Techniques: Stable Transfection, shRNA, Migration, Expressing, Knockdown, Western Blot, Immunofluorescence, CCK-8 Assay, Staining, Flow Cytometry, Transwell Invasion Assay, Transfection, Plasmid Preparation

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Co-expression of COMMD1 and COMMD7 Suppressed the Expression of the NF-κB Signaling Pathway Nanog + HCSCs were transfected with empty vector, pcDNA3.1-COMMD1 alone, pcDNA3.1-COMMD1 + pcDNA3.1-COMMD7, or pcDNA3.1-COMMD1 + sh-COMMD7, respectively. (A) The expression of COMMD1 and COMMD7 was determined using real-time qPCR analysis. Western blotting analysis was performed to detect the expression of (B) COMMD7, COMMD1, and HNF4α, as well as (C) NF-κB p65 in the nucleus and cytoplasm, respectively. (D and E) Immunofluorescence images of HNF4α (D) and NF-κB (E). Scale bars, 50 μm. ***p < 0.001.

Article Snippet: For immunofluorescence, cells were fixed in 4% paraformaldehyde and then washed with PBS for 5 min. Next, cells were incubated with primary antibody against COMMD7, COMMD1, p65, or HNF4α at 4°C overnight, followed by incubation with FITC-labeled goat against secondary antibody (Beyotime Institute of Biotechnology).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immunofluorescence

Journal: Molecular Therapy Oncolytics

Article Title: COMMD7 Regulates NF-κB Signaling Pathway in Hepatocellular Carcinoma Stem-like Cells

doi: 10.1016/j.omto.2018.12.006

Figure Lengend Snippet: Overexpression of PIAS4 Influenced the Effect of COMMD7 Knockdown in Nanog + HCSCs (A and B) The expression of PIAS4 was determined using real-time qPCR (A) and western blotting (B) analysis of the protein level of CXCL12, CXCL2, and PIAS4 in Nanog + HCSCs after COMMD7 knockdown. Then the COMMD7-silenced Nanog + HCSCs were transfected with empty vector or pcDNA3.1-PIAS4. (C) The mRNA expression of PIAS4 after PIAS4 overexpression. (D) Western blotting analysis of the protein expression of NEMO, p65, HNF4α, and PIAS4. The proliferative ability, apoptotic rate, migration, and invasion were evaluated using (E) CCK-8 assay, (F) flow cytometry assay, and (G) wound healing and transwell invasion assay in Nanog + HCSCs, respectively. *p < 0.05; ***p < 0.001 versus vector or sh-COMMD7.

Article Snippet: For immunofluorescence, cells were fixed in 4% paraformaldehyde and then washed with PBS for 5 min. Next, cells were incubated with primary antibody against COMMD7, COMMD1, p65, or HNF4α at 4°C overnight, followed by incubation with FITC-labeled goat against secondary antibody (Beyotime Institute of Biotechnology).

Techniques: Over Expression, Knockdown, Expressing, Western Blot, Transfection, Plasmid Preparation, Migration, CCK-8 Assay, Flow Cytometry, Transwell Invasion Assay

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Short-term ex vivo culture of hepatic cells from e18.5 (embryonic day) HNF4α-null liver restores polarity and partially rescues expression of the E-cadherin gene. (A) Breeding scheme used to generate null (HNF4αfl/− AlfpCreTg/0) and littermate control (HNF4aαfl/+ AlfpCreTg/0). (B) Double immunofluorescence for tight junction protein 1 (TJP1/ZO1, green), and HNF4α (red) with nuclei visualized by DAPI (blue) staining, carried out at 3 and 7 days of culture. The “3 days” photomicrographs have bars to show vertical distribution of the fluorescence signals through the culture (z-stack). Immunostaining for HNF4α is intense in the control cells at 7 days, and a single stained nucleus has been captured for the null culture. (C) E-Cadherin transcripts, for control (open bars) and HNF4α-null (filled bars) e18.5 livers (left) and e18.5 three day cultures of hepatic cells (right).

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Ex Vivo, Expressing, Control, Immunofluorescence, Staining, Fluorescence, Immunostaining

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Morphology and cell polarity of bipotential mouse embryonic liver (BMEL) cell lines grown in basal or differentiation-inducing culture conditions is independent of HNF4α genotype. (A) Structure of the floxed and null alleles of HNF4α and Southern blot analysis of HNF4α-floxed BMEL cell lines stably transfected with vectors expressing GFP (green fluorescent protein) alone (GFP, Control) or a GFPnlsCre-fusion protein (GFPCre, null). Positions of BamHI sites (B), loxP sites (●), exons (■) and the probe (□) are represented. The HNF4α-floxed and -null alleles yield bands of 3.4 and 5.6 Kb, respectively. (B) Phase-contrast micrographs of cells grown under basal (proliferation) conditions and in Matrigel. (C) Immunofluorescent staining of tight junction protein 1 (TJP1) and E-cadherin.

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Southern Blot, Stable Transfection, Transfection, Expressing, Control, Staining

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: BMEL cells, whether they can express functional HNF4α or not, have the potential to form functional bile canalilculi. Frames illustrate loading of bile canalicular pockets with fluorescein during 0–12 min at 37 °C after addition of FDA, using the null clone Cre14 and control GFP2. At time 0 fluorescence is in the cytoplasm, and progressively disappears as the vesicles become fluorescent. Individual fields at successive times, with arrows identifying the evolution of fluorescence of bile canaliculi, that can be seen as spaces between the confluent cells in the phase contrast image (right). Images at T0 were captured as rapidly as possible after FDA was added to the cultures. T1 was 1 min later for Cre14 (Fields 1 and 2), and 2 min later for GFP2. T2 corresponds to 2 min for Cre14 and to 11 min for GFP2. (The rates of exchange differ from one field to another in a given dish; the differences in timing are not considered significant.)

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Functional Assay, Control, Fluorescence

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Control and HNF4α-null BMEL cells participate in liver regeneration in the Alb-uPA/SCID mouse, forming polarized, differentiated bile ducts. (A) Liver sections of Alb-uPA/SCID (Albu-min-urokinase Plasminogen Activator trangene/Severe Combined Immunodeficiency) mice injected with HNF4α control BMEL cells. Immunohistochemistry (brown) of serial adjacent sections for GFP and for the bile duct-specific cytokeratins CK19 and CK7, and for E-cadherin, expressed in both hepatocytes and cholangiocytes. Two sections from a different area, stained, respectively, with GFP, highlighting both a cluster of BMEL hepatocytes and a bile duct. HNF1β nuclear staining is observed as expected in hepatocytes and especially in bile duct cells. (B) Alb-uPA/SCID mice transplanted with HNF4α-null BMEL cells. Four serial sections showing BMEL derived bile ducts stained with GFP, CK19, CK7 and E-cadherin. Two serial sections stained, respectively, with GFP and HNF1β.

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Control, Injection, Immunohistochemistry, Staining, Derivative Assay

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Gene expression patterns in HNF4α-null and control liver and primary hepatic cell cultures. Steady-state RNA concentrations in: (A) control (open bars) and HNF4α-null (filled bars) embryonic livers during development, and (B) 3 day primary cultures from e18.5 embryonic livers, as measured by real-time PCR. Cholangiocyte markers include a cytokeratin (CK19), a surface receptor (c-kit) and a transcription factor (HNF6). Hepatocyte markers include a serum protein (AFP, alpha-feto protein), genes required for lipid transport (Apoa1, apolipoprotein; Apoa4), and amino acid (Mat1a, methionine adenosyl transferase; TAT; tyrosine aminotransferase) and carbohydrate (Pck1, phospho(enol)pyruvate carboxykinase) metabolism. Values were corrected to 18s and are expressed relative to the mean value for control livers at e14.5 (±standard deviation, n = 4), or relative to control livers (standard deviation, n =≥3) in primary culture. Histograms are means of ≥3 livers or cultures from ≥3 different livers. Statistical comparisons of the effect of genotype on RNA concentrations in embryonic liver tissue were performed using unpaired, 2-tailed t-tests assuming equal variance. *≤60.05, ** ≤0.01.

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Disruption of allele HNF4αtm1.1Gonz in the developing mouse liver leads to defects in hepatic morphogenesis. Breeding scheme used to generate null (HNF4αfl/− AlfpCreTg/0) and littermate control (HNF4αfl/+ AlfpCreTg/0) embryos is depicted in Fig. 1A. (A) Immunohistochemistry for HNF4α on embryonic mouse liver (10 µm sections) at e14.5 and e18.5 stages of development. HNF4α positive nuclei stain brown. In occasional 18.5 day samples, clonal proliferation of cells that have escaped gene inactivation was observed. (B) Immunocytology of TJP1 (bar = 100 µm) and E-cadherin in control and HNF4α-null mouse liver at e12.5 (10×) and e18.5 (20×).

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Disruption, Control, Immunohistochemistry, Staining

Journal:

Article Title: Morphogenetic competence of HNF4?-deficient mouse hepatic cells ✩

doi: 10.1016/j.jhep.2008.04.024

Figure Lengend Snippet: Gene expression patterns in HNF4α-null embryonic liver indicate multiple metabolic defects and late-onset cellular responses to stress and hypoxia. Steady-state RNA concentrations in control (open bars) and HNF4α-null (filled bars) embryonic livers during development as measured by real-time PCR. First row: an essential metabolic enzyme of the liver (FAH, fumerylacetoacetate hydrolase) and markers of cellular stress (c-Fos), hypoxia (Epo, Erythropoietein) and DNA damage (Ddit3, DNA damage inducible). Second row: markers of the major cell lineages in the developing liver: hepatoblasts (HNF1β, Osmr, oncostatin M receptor), hematopoietic stem cells (Osm) and angioblasts (Vegfc, vascular endothelial growth factor). Values were corrected to 18s and are expressed relative to the mean value for control livers at e14.5 (±standard deviation. n = 4). See legend to Fig. 5 for statistical analysis.

Article Snippet: We thank all members of the Plateforme d’Imagerie Dynamique, Pasteur Institute, and particularly Emmanuelle Perret for their help with image acquisition, Francois Tronche and Lionel Gresh for the Alfp-Cre mice, Francey Sladek and Doris Cassio for HNF4α and ZO1 antibodies, respectively, Margaret Buckingham, and all members of the Unité de Génétique de la Différenciation for advice and constructive criticism.

Techniques: Gene Expression, Control, Real-time Polymerase Chain Reaction, Standard Deviation