commd7  (Novus Biologicals)

Journal: International Journal of Medical Sciences

Article Title: ZNF460-regulated COMMD7 Promotes Acute Myeloid Leukemia Proliferation Via the NF-κB Signaling Pathway

doi: 10.7150/ijms.80047

Figure Lengend Snippet: COMMD7 is highly expressed and promotes proliferation of AML cells. The mRNA and protein levels of COMMD7 in NB4, KG1a, THP1, U937, MV-411 and PBMCs (A). Different COMMD7 shRNAs were employed into KG1a and U937 cells. Knock-down efficiency was detected by qRT-PCR (B) and western blotting (C). Knockdown of COMMD7 inhibited KG1a and U937 cells proliferation. Cell viability was analyzed by the CCK-8 assay (D).

Article Snippet: The antibodies employed were as follows: Cleaved-Caspase-3 (#9961,Cell Signaling Technology, USA), PARP (#A5037, Bimake, USA), NF-κB p50/105 (#A5600, Bimake), Bcl2 (#A5010, Bimake), Phospho-CDK1 (#R26267, ZENBIO,China), Caspase3 (#9622, Cell Signaling Technology), NF-κB p65 (#A5075,Bimake), Cleaved-PARP (#A5034, Bimake), Bax (#5023, Cell Signaling Technology), Cyclin B1 (#R23324, ZENBIO, China), MMP9 (#R380831, ZENBIO), ZNF460 (#TD4628, Abmart, China), p38 (#8690, Cell Signaling Technology), COMMD7 (#GTX112076, GeneTex, USA), p21 (#A5163, Bimake), CDK1 (#R23884, ZENBIO), c-Myc (Wanleibio, China), p53 (#A5722, Bimake).

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, CCK-8 Assay

Journal: International Journal of Medical Sciences

Article Title: ZNF460-regulated COMMD7 Promotes Acute Myeloid Leukemia Proliferation Via the NF-κB Signaling Pathway

doi: 10.7150/ijms.80047

Figure Lengend Snippet: Knockdown of COMMD7 promotes apoptosis and G2/M arrest via the NF-κB pathway. Apoptosis rates of KG1a and U937 were measured by flow cytometry by using annexin V/PI kit (A). Expression of apoptosis-related proteins was tested by western blotting (B). Cell cycle was determined by flow cytometry (C). Western blotting was employed to assayed the cell cycle-associated proteins (D). NF-κB pathway-related protein expression was measured by immunoblotting (E).

Article Snippet: The antibodies employed were as follows: Cleaved-Caspase-3 (#9961,Cell Signaling Technology, USA), PARP (#A5037, Bimake, USA), NF-κB p50/105 (#A5600, Bimake), Bcl2 (#A5010, Bimake), Phospho-CDK1 (#R26267, ZENBIO,China), Caspase3 (#9622, Cell Signaling Technology), NF-κB p65 (#A5075,Bimake), Cleaved-PARP (#A5034, Bimake), Bax (#5023, Cell Signaling Technology), Cyclin B1 (#R23324, ZENBIO, China), MMP9 (#R380831, ZENBIO), ZNF460 (#TD4628, Abmart, China), p38 (#8690, Cell Signaling Technology), COMMD7 (#GTX112076, GeneTex, USA), p21 (#A5163, Bimake), CDK1 (#R23884, ZENBIO), c-Myc (Wanleibio, China), p53 (#A5722, Bimake).

Techniques: Knockdown, Flow Cytometry, Expressing, Western Blot

Journal: International Journal of Medical Sciences

Article Title: ZNF460-regulated COMMD7 Promotes Acute Myeloid Leukemia Proliferation Via the NF-κB Signaling Pathway

doi: 10.7150/ijms.80047

Figure Lengend Snippet: ZNF460 interacts with COMMD7 and promotes AML cell proliferation. Potential transcription factors of COMMD7 promoter were projected through linking UCSC database with JASPAR website (A). Correlation between ZNF460 and COMMD7 was analyzed by GEPIA (B). Expression of ZNF460 in normal and AML groups (C). Expression of ZNF460 in AML cell lines was assessed by qRT-PCR and western blotting (D). Protein extracted from KG1a and U937 cells was co-immunoprecipitated with anti-ZNF460 antibody and blotted with anti-ZNF460 and anti-COMMD7 antibodies (E). Knockdown efficiency of ZNF460 in KG1a and U937 cells and expression of COMMD7 after knockdown of ZNF460 were detected by qRT-PCR (F) and western blotting (G). CCK-8 assays were used to assess cell proliferation (H).

Article Snippet: The antibodies employed were as follows: Cleaved-Caspase-3 (#9961,Cell Signaling Technology, USA), PARP (#A5037, Bimake, USA), NF-κB p50/105 (#A5600, Bimake), Bcl2 (#A5010, Bimake), Phospho-CDK1 (#R26267, ZENBIO,China), Caspase3 (#9622, Cell Signaling Technology), NF-κB p65 (#A5075,Bimake), Cleaved-PARP (#A5034, Bimake), Bax (#5023, Cell Signaling Technology), Cyclin B1 (#R23324, ZENBIO, China), MMP9 (#R380831, ZENBIO), ZNF460 (#TD4628, Abmart, China), p38 (#8690, Cell Signaling Technology), COMMD7 (#GTX112076, GeneTex, USA), p21 (#A5163, Bimake), CDK1 (#R23884, ZENBIO), c-Myc (Wanleibio, China), p53 (#A5722, Bimake).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Immunoprecipitation, Knockdown, CCK-8 Assay

Journal: Blood Advances

Article Title: CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

doi: 10.1182/bloodadvances.2020004112

Figure Lengend Snippet: Effect of COMMD7 KO on megakaryopoiesis. (A) Real-time PCR analysis of COMMD7 RNA expression in COMMD7 CRISPR-treated day 13 MKs. Data are normalized to GAPDH (3 independent cords per group). (B) Western blot of COMMD7 protein in platelets from 3 healthy donors and in control and COMMD7 KO MKs from 3 independent cords per group. β-actin (ACTB) was used as loading control. (C) Percentage of viable negative control or COMMD7 KO cells on day 13 of culture. (D) Proplatelet formation on day 13 MKs plated overnight on fibrinogen and stained with Phalloidin 488. Shown are representative images of 10 random images taken from each of 3 cords. The percentage of control or COMMD7 KO MKs making proplatelets on day 13 was counted blinded to group and summarized in the bar graph on the right. (E) Flow cytometry analysis of MK maturation markers on day 13 control or COMMD7 MKs. Viable MKs were gated as in Figure 2B. Shown is the mean ± SEM of the percent of cells (y-axis) expressing MK maturation markers. Student t test: *P < .05; **P < .01.

Article Snippet: Proteins were detected with antibodies, including calcium diacylglycerol-guanine exchange factor 1 (CalDAG-GEF1) ( RASGRP2 ) (Genetex #GTX108616), αIIb (Santa Cruz #166599), GP6 (R&D Systems #AF3627), B2M (Abcam #Ab75853), COMMD7 (Novus Biologicals #NBP2-58399), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz # 47724), and phospho-protein kinase C (pPKC) substrates (Cell Signaling #6967).

Techniques: Real-time Polymerase Chain Reaction, RNA Expression, CRISPR, Western Blot, Control, Negative Control, Staining, Flow Cytometry, Expressing

Journal: Blood Advances

Article Title: CRISPR-edited megakaryocytes for rapid screening of platelet gene functions

doi: 10.1182/bloodadvances.2020004112

Figure Lengend Snippet: Effect of COMMD7 KO on platelet functional responses in MKs. (A-B,D-F) Negative control or COMMD7 KO day 13 MKs were kept at baseline or stimulated with convulxin (2 μg/mL) or thrombin (1 U/mL) for 3 or 10 minutes as indicated and analyzed by flow cytometry. Viable MKs were gated as in Figure 2B. Shown is the normalized mean ± SEM of percent positive cells for PAC-1 (A), labeled fibrinogen (B), surface P-selectin (D-E) , or surface CD63 (F) (3-5 independent cords per group). (C) Mean ± SEM of the percentage of control or CRISPR KO MKs spread after 1 hour on collagen or fibrinogen. Shown are samples from 3 cords with each sample plated in duplicate wells; >20 fields and >200 MKs were scored (blinded to treatment) per sample. Paired Student t tests: *P < .05; **P < .01.

Article Snippet: Proteins were detected with antibodies, including calcium diacylglycerol-guanine exchange factor 1 (CalDAG-GEF1) ( RASGRP2 ) (Genetex #GTX108616), αIIb (Santa Cruz #166599), GP6 (R&D Systems #AF3627), B2M (Abcam #Ab75853), COMMD7 (Novus Biologicals #NBP2-58399), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz # 47724), and phospho-protein kinase C (pPKC) substrates (Cell Signaling #6967).

Techniques: Functional Assay, Negative Control, Flow Cytometry, Labeling, Control, CRISPR

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: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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 COMMD1 Partially Reversed the Function of COMMD7 in Nanog + HCSCs 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) CCK-8 assay was performed to evaluate proliferative ability. (B) Flow cytometry assay was performed to quantify the apoptotic rate. (C) Wound healing and transwell invasion assay were used to determine cell migration and invasion ability. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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

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: Bioinformatics Analysis of Gene Expression Patterns Involved in NF-κB Signaling Pathway in Nanog + HCSCs (A) Heatmap generated by hierarchical clustering of differentially downregulated or upregulated expressed genes in Nanog + HCSCs after COMMD7 knockdown. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of DEGs. (B) For upregulated DEGs, the top 10 enriched pathways are shown. (C) For downregulated DEGs, the top 10 enriched pathways are shown.

Article Snippet: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

Techniques: Gene Expression, Generated, Knockdown

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: Next, the membranes were blocked in 5% nonfat milk dissolved in Tris-buffered saline with Tween 20 (TBST) solution for 1 h at room temperature followed by incubation with primary antibodies against Nanog (1:1,000, ab153419; Abcam), COMMD7 (1:2,000, #9742; Cell Signaling), COMMD1 (1:1,000, #24640; SAB), NF-κB p65 (1:1,500, 19553-1-AP; Proteintech), HNF4α (1:1,000, #2947; Cell Signaling), NEMO (1:3,000, #23870; SAB), CXCL12 (1:1,000, #36120; SAB), CXCL2 (1:4,000, #24681; SAB), PIAS4 (1:2,500, #8709; Cell Signaling), histone H3.1 (1:2,000, #9265; Cell Signaling), β-actin (1:1,000, #5762; Cell Signaling), and GAPDH (1:10,000, 10494-1-AP; Proteintech) at 4°C overnight.

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

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