Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: The effect of curcumin on cell proliferation and apoptosis in ovarian cancer cells. Cell viability ( A ), colony-formation ability ( B ), apoptosis ( C ), and protein levels of c-caspase-3, PCNA and Bax ( D ) were detected in SKOV3 and A2780 cells after exposure to various doses of curcumin. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques:

Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: The influence of circ-PLEKHM3 on the proliferation and apoptosis in curcumin-treated ovarian cancer cells. A Circ-PLEKHM3 expression was examined in tumor and normal tissues ( n = 35). B Circ-PLEKHM3 level was measured in SKOV3, A2780 and IOSE-80 cells. C Circ-PLEKHM3 abundance was examined in SKOV3 and A2780 cells after stimulation of various concentrations of curcumin. D Circ-PLEKHM3 level was detected in cells with transfection of oe-circ-PLEKHM3 or vector. Colony-formation ability ( E ), apoptosis ( F ), and protein levels of c-caspase-3, PCNA and Bax ( G ) were examined in cells with transfection of oe-circ-PLEKHM3 or vector under the treatment of curcumin or DMSO. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: The influence of circ-PLEKHM3 and curcumin on tumor growth in ovarian cancer. A2780 cells transfected with oe-circ-PLEKHM3 or vector were injected into nude mice, and next mice were subjected to curcumin or DMSO. Tumor volume ( A ), weight ( B ), circ-PLEKHM3 expression ( C ), and protein abundances of c-caspase-3, PCNA and Bax ( D ) were detected in each group. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques: Transfection, Plasmid Preparation, Injection, Expressing

Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: Circ-PLEKHM3 sponged miR-320a. A MiR-320a expression was detected in tumor and normal tissues ( n = 35). B MiR-320a level was measured in SKOV3, A2780 and IOSE-80 cells. C The linear correlation of miR-320a and circ-PLEKHM3 in ovarian cancer tissues was analyzed. D MiR-320a abundance was examined in SKOV3 and A2780 cells after exposure to various concentrations of curcumin. E The binding site of circ-PLEKHM3 and miR-320a was predicted via starBase. F Luciferase activity was detected in 293 T cells with transfection of circ-PLEKHM3 wt or mut and miR-320a mimic or mimic NC. G Circ-PLEKHM3 and miR-320a abundances were detected after RIP. H MiR-320a abundance was examined in SKOV3 and A2780 cells transfected with mimic NC, miR-320a mimic, inhibitor NC or miR-320a inhibitor. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques: Expressing, Binding Assay, Luciferase, Activity Assay, Transfection

Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: SMG1 was a target of miR-320a. A and B SMG1 level was measured in tumor and normal tissues ( n = 35). C and D SMG1 level was examined in SKOV3, A2780 and IOSE-80 cells. E The linear correlation of miR-320a and SMG1 in ovarian cancer tissues was analyzed. F and G SMG1 abundance was detected in SKOV3 and A2780 cells after exposure to various concentrations of curcumin. H The binding site of miR-320a and SMG1 was predicted via starBase. I Luciferase activity was detected in 293 T cells transfected with SMG1 3’UTR wt or mut and miR-320a mimic or mimic NC. J The interaction between miR-320a and SMG1 was analyzed by RIP assay. K-L SMG1 abundance was measured in SKOV3 and A2780 cells transfected with si-NC or si-SMG1. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection

Journal: Journal of Ovarian Research

Article Title: Curcumin inhibits ovarian cancer progression by regulating circ-PLEKHM3/miR-320a/SMG1 axis

doi: 10.1186/s13048-021-00916-8

Figure Lengend Snippet: The influence of miR-320a and SMG1 on ovarian cancer progression under curcumin exposure. A and B SMG1 expression was detected in SKOV3 and A2780 cells with transfection of inhibitor NC, miR-320a inhibitor, miR-320a inhibitor + si-NC or si-SMG1. Colony-formation ability ( C) , apoptosis ( D ), and protein levels of c-caspase-3, PCNA and Bax ( E ) were measured in SKOV3 and A2780 cells with transfection of inhibitor NC, miR-320a inhibitor, miR-320a inhibitor + si-NC or si-SMG1 after exposure to curcumin. * P < 0.05

Article Snippet: Ovarian cancer cell lines [SKOV3 (Cat. No. CL-0215) and A2780 (Cat. No. CL-0013) cells] and human embryonic kidney 293 T cells (Cat. No. CL-0005) were provided by Procell Life Science Technology (Wuhan, China).

Techniques: Expressing, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: AGEs upregulate β-catenin protein level and activate its transcriptional activity. A The enrichment analysis of KEGG pathways of differentially expressed genes (DEGs) induced by AGEs. B Heatmap of Representative DEGs enriched in the Wnt signaling pathway. C qPCR analysis of representative DEGs enriched in Wnt signaling pathway. n = 4. D HUVECs were treated by AGEs (100 μg/mL) for different times and β-catenin protein level was measured by western blot. n = 5. E qPCR analysis of β-catenin mRNA level in HUVECs with AGEs (100 μg/mL) treatment for different times. n = 4. F Heatmap of Wnt signaling upstream genes that affect β-catenin protein level, including Wnt receptors, downstream genes of them and destruction complex genes. G qPCR analysis of Wnt signaling upstream genes mRNA levels in HUVECs with AGEs (100 μg/mL) treatment. n = 4. H HUVECs were treated by AGEs (100 μg/mL) for different times. Proteins of nuclear fractions were isolated and then β-catenin protein was detected by western blot. Lamin B1 was used as an internal control for nuclear proteins. n = 5. NL indicates the nuclear lysate. I HUVECs were treated by AGEs (100 μg/mL) for 8 h, and then stained for β-catenin (red). The nuclei were stained with DAPI (blue). The line charts show the mean fluorescence intensity (MFI) of the distance in the images from α to ω in arbitrary units (AU). Scale bar indicates 50 μm. J 293T cells were transfected with luciferase constructs containing TCF/LEF response element (RE) and cultured for 24 h followed by AGEs treatment. Dual-Luciferase Reporter Assay was performed to detect the luciferase reporter activities the and luminescence was normalized to Renilla. n = 8. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Activity Assay, Western Blot, Isolation, Control, Staining, Fluorescence, Transfection, Luciferase, Construct, Cell Culture, Reporter Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: β-catenin increases the expression of KDR by upregulating NANOG under AGEs treatment. A , B 293T cells were transfected with KDR promoter luciferase construct followed by AGEs stimulation in the presence or absence of 20 μM ICG-001 for 24 h ( A ) or β-catenin siRNA ( B ). The Dual-Luciferase Reporter Assay was used to detect promoter activity, and luminescence was normalized to Renilla. n = 5. C 293T cells were co-transfected with KDR promoter luciferase construct and β-catenin-overexpression plasmid. After 24 h. The Dual-Luciferase Reporter Assay was performed to detect promoter activity, and luminescence was normalized to Renilla. n = 5. D Schematic diagram of the predicted binding region of β-catenin to the KDR promoter DNA sequence (upper panel) and the PCR amplification product location of 8 primers for the predicted binding element (lower panel). IBS software was used as a drawing tool . E The binding of β‐catenin to the KDR promoter in response to AGEs treatment was determined in HUVECs by ChIP assay and quantified by PCR. Experiments were repeated three times with similar results and one representative result is shown. F Venn diagram showing overlapping genes between transcriptional factors of KDR and Wnt/β-catenin target genes. G - I HUVECs were treated with AGEs (100 μg/ml) for different times and the mRNA levels of GATA2/SP1/NANOG were detected by qPCR. n = 5 to 7. J , K HUVECs were pretreated with ICG-001 (20 μM) ( J ) or β-catenin siRNA ( K ) followed by AGEs (100 μg/ml) stimulation and NONOG mRNA levels were detected by qPCR. n = 5. L siRNA targeting NANOG was transfected into HUVECs followed by AGEs (100 μg/ml) treatment and KDR mRNA levels were detected by qPCR, respectively. n = 4. M 293T cells were co-transfected with KDR promoter luciferase construct, β-catenin-overexpression plasmid and NONOG siRNA. After 48 h, the Dual-Luciferase Reporter Assay was conducted to detect KDR promoter activity, and luminescence was normalized to Renilla. n = 6. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Expressing, Transfection, Luciferase, Construct, Reporter Assay, Activity Assay, Over Expression, Plasmid Preparation, Binding Assay, Sequencing, Amplification, Software

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: AGEs mediate β-catenin phosphorylation and Y142 is the key site of phosphorylation. A HUVECs were treated with AGEs (100 μg/ml) for different times, and then serine/threonine phosphorylation and tyrosine phosphorylation level of β-catenin were detected by western blot. One representative cropped blot of three independent experiments is shown. B Schematic diagram of constructing the three types of β-catenin multi-site mutant (Y to F) plasmids. Vertical line in green means tyrosine (Y) while line in red means the site is mutated to phenylalanine (F). Illustrated by IBS software. C β-catenin multi-site mutant (Y to F) plasmid was transfected into 293T cells followed by AGEs treatment, and then tyrosine phosphorylation level of β-catenin was detected by western blot. n = 4. One representative cropped blot of four independent experiments is shown and the phosphorylation protein level is calculated relative to each group without AGEs treatment, defined as “1”. D Schematic diagram of constructing the four types of β-catenin single-site mutants (Y to F) plasmids. Vertical line in green means tyrosine (Y) while line in red means the site is mutated to phenylalanine (F). Illustrated by IBS software. E The β-catenin single-site mutant (Y to F) plasmid was transfected into 293T cells followed by AGEs treatment, and then tyrosine phosphorylation level of β-catenin was detected by western blot. n = 4. One representative cropped blot of four independent experiments is shown and the phosphorylation protein level is calculated relative to each group without AGEs treatment, defined as “1”. F Amino acid sequence alignment of β-catenin Y142 residues among β-catenin homologs from different species. Y142 residue in various species is colored in red. G HUVECs were treated with AGEs (100 μg/ml) for 1 h and then phosphorylation of β-catenin Y142 was measured by western blot. n = 5. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Phospho-proteomics, Western Blot, Mutagenesis, Software, Plasmid Preparation, Transfection, Sequencing, Residue

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: β-catenin Y142 phosphorylation accounts for AGE-induced β-catenin transcriptional activity and angiogenesis. A After transfection with β-catenin WT or Y142F-overexpression adenovirus for 48 h, HUVECs were stimulated by AGEs (100 μg/mL) for 8 h. Proteins of the nuclear and whole cell fractions were isolated and then β-catenin protein level was detected by western blot, respectively. n = 6. NL indicates nuclear lysate; WCL indicates whole cell lysate. B - D 293T cells were co-transfected with β-catenin WT or Y142F-overexpression adenovirus and TCF/LEF response element (RE) ( B ), KDR promoter ( C ) or HDAC9 promoter ( D ) luciferase constructs followed by AGEs stimulation (100 μg/ml). The Dual-Luciferase Reporter Assay was conducted to detected luciferase reporter activities, and luminescence was normalized to Renilla. n = 5–6. E – G After transfection with β-catenin WT or Y142F-overexpression adenovirus for 48 h, HUVECs were stimulated with AGEs (100 μg/ml) for 24 h followed by the CCK8 assay ( E ), Transwell assay ( F ), and tube formation assay ( G ) to evaluate the OD value, migrated cells number and tube length, branching points, respectively. n = 3 to 6, scale bar indicates 100 or 200 μm. H Matrigel containing HUVEC transfected Ad-WT or Ad-Y142F in the presence or absence of AGEs (100 μg/mL) treatment was injected subcutaneously into mouse. A week later, plugs were harvested. Ulex europaeus agglutinin I (UEAI, Red) was used to stain HUVECs and HUVECs-formed blood vessels were measured. n = 3, scale bar indicates 100 μm. Ad indicates adenovirus overexpressing the corresponding protein. I Aortic rings were transfected with Ad-WT or Ad-Y142F, treated with or without AGEs (100 μg/mL) for 6 days and then stained with isolectin B4 (IB4; green). The mean number of sprout (indicated by red dot) from aortic ring was counted. n = 5, scale bar indicates 100 μm. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Phospho-proteomics, Activity Assay, Transfection, Over Expression, Isolation, Western Blot, Luciferase, Construct, Reporter Assay, CCK-8 Assay, Transwell Assay, Tube Formation Assay, Injection, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: VEGFR1 isoform 5 accounts for β-catenin Y142 phosphorylation. A Venn diagram showing overlapping genes between the kinases of β-catenin Y142 predicted in silico from the GPS database and AGE-upregulated kinases from DEGs of transcriptomic. B Schematic diagram of the 7 isoforms of VEGFR1. Detailed information was obtained from the Uniprot database. C HUVECs were stimulated by AGEs (100 μg/mL) for 1 h, and the full-length VEGFR1 levels were measured by western blot. n = 5. D HUVECs were treated with AGEs (100 μg/mL) for 1 h and then Co-IP assay was performed with β-catenin antibody to analyze the interaction between β-catenin and the full-length VEGFR1. Experiments were repeated three times with similar results and one representative result is shown. E HUVECs were stimulated by AGEs (100 μg/mL) for 1 h, and the VEGFR1 isoform5, isoform6 and isoform7 protein levels were measured by western blot. n = 8. F HUVECs were stimulated by AGEs (100 μg/mL) for 1 h and then Co-IP assay was performed with β-catenin antibody to analyze the interaction between β-catenin and isoform5/6/7. Experiments were repeated six times with similar results and one representative result is shown. G The HA-tagged VEGFR1 isoform5, isoform6 or isoform7 plasmid was transfected into 293T cells followed by AGEs treatment for 1 h, and then Co-IP assay was performed with HA antibody to analyze the interaction between endogenic β-catenin and ectogenic VEGFR1 isoform5/6/7. Experiments were repeated three times with similar results and one representative result is shown. H The Flag-tagged β-catenin-overexpressing plasmid was co-transfected with HA-tagged VEGFR1 isoform5, isoform6 or isoform7 plasmid into 293T cells followed by AGEs treatment for 1 h, and then Co-IP assay was performed with HA antibody to analyze the interaction between ectogenic β-catenin and these three isoforms of VEGFR1. Experiments were repeated three times with similar results and one representative result is shown. I HUVECs were transfected with siRNA targeting VEGFR1 isoform5 followed by AGEs (100 μg/mL) for 1 h and the phosphorylation level of β-catenin Y142 was quantified by western blot. n = 5. J 293T cells were transfected with VEGFR1 isoform5-overexpression plasmid followed by AGEs (100 μg/mL) for 1 h and the phosphorylation level of β-catenin Y142 was quantified by western blot. n = 5. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Phospho-proteomics, In Silico, Western Blot, Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Over Expression

Journal: Cell Communication and Signaling : CCS

Article Title: Endothelial β-catenin upregulation and Y142 phosphorylation drive diabetic angiogenesis via upregulating KDR / HDAC9

doi: 10.1186/s12964-024-01566-1

Figure Lengend Snippet: Bioymifi acts as an inhibitor of VEGFR1–β-catenin interaction and blocks AGE-induced angiogenesis. A The bound conformation of the VEGFR1 isoform5 and β-catenin was predicted by the ZDOCK algorithm. VEGFR1 isoform5 is displayed in yellow, and β-catenin is displayed in green. B Inhibitor binding pockets on the β-catenin protein mode were predicted using MOE-Site Finder plug-ins. C 293T cells were co-transfected with the Flag-tagged β-catenin-overexpression plasmid and HA-tagged VEGFR1 isoform5-overexpression plasmid followed by Bioymifi treatment with different concentrations in the presence of AGEs (100 μg/mL) and then Co-IP assay was performed with HA tag antibody to analyze the interaction between β-catenin and VEGFR1 isoform5. Experiments were repeated three times with similar results and one representative result is shown. D The molecular structure of Bioymifi. E Computational model of the interaction between Bioymifi and the VEGFR1 isoform5-β-catenin complex generated by AutoDock Vina and the key residues for the interaction between Bioymifi and β-catenin. F A 2D docking model showing the interactions between Bioymifi and β-catenin using MOE software. G HUVECs were pretreated with 2 μM Bioymifi for 2 h and then stimulated with AGEs (100 μg/ml) for 1 h and then β-catenin Y142 phosphorylation was measured. n = 6. H - J 293T cells were transfected with TCF/LEF response element (RE) ( H ), KDR promoter ( I ) or HDAC9 promoter ( J ) luciferase constructs followed by AGEs (100 μg/ml) stimulation in the presence or absence of 2 μM Bioymifi. The Dual-Luciferase Reporter Assay was conducted to detected luciferase reporter activities, and luminescence was normalized to Renilla. n = 5. ( K - M ) HUVECs were treated with AGEs (100 μg/mL) for 24 h in the presence of 2 μM Bioymifi, and then the CCK8 assay ( K ), Transwell assay ( L ), and tube formation assay ( M ) were carried out to evaluate the OD value, migrated cell number and the tube length, branching points, respectively. n = 4 to 5, scale bar indicates 100 or 200 μm. N Aortic rings were treated with or without AGEs (100 μg/mL) for 6 days in the presence of 2 μM Bioymifi and then stained with isolectin B4 (IB4; green). The mean number of sprout (indicated by red dot) from aortic ring was counted. n = 5, scale bar indicates 100 μm. O Schematic diagram illustrating the function of β-catenin in AGEs-induced diabetic angiogenesis. Data are shown as Mean ± SD . * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: HUVECs and 293T cells were purchased from ScienCell Research Laboratories (CA, USA, Cat# 8000) and Procell Life Science & Technology (Wuhan, China, Cat# CL-0005), respectively.

Techniques: Binding Assay, Transfection, Over Expression, Plasmid Preparation, Co-Immunoprecipitation Assay, Generated, Software, Phospho-proteomics, Luciferase, Construct, Reporter Assay, CCK-8 Assay, Transwell Assay, Tube Formation Assay, Staining