Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Results of flow cytometry, western blotting, and qPCR for determining the macrophage polarization status and the expression of proangiogenic factors. (A) Representative flow cytometry histogram showing the fluorescence intensity of 6 macrophage polarization markers. (B) Bar charts showing the MFIs of macrophage polarization markers ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (C) Representative western blot showing the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 (normalized to β‐Actin expression) in primary macrophages. (D) Densitometric analyses of the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (E) qPCR results showing the relative mRNA expression of iNOS , TNF‐α , IL‐6 , VEGFA , IGF1 , EGR1 , IL‐10 , and MMP9 in primary macrophages ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (F) Representative flow cytometry histogram showing the fluorescence intensity of macrophage polarization markers. (G) Bar charts showing the MFIs of macrophage polarization markers ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (H) Representative western blot showing the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 (normalized to β‐Actin expression) in TDMs. (I) Densitometric analyses of the relative levels of iNOS, TNF‐α, IL‐6, VEGFA, IGF1, EGR1, IL‐10, and MMP9 ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). (J) qPCR results showing the relative mRNA expression of iNOS , TNF‐α , IL‐6 , VEGFA , IGF1 , EGR1 , IL‐10 , and MMP9 in TDMs ( n = 3; one‐way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. MFI, mean fluorescence intensity; TDMs, THP‐1‐derived macrophages; TEMs, Tie2‐expressing monocytes/macrophages; TNMs, Tie2‐negative monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Flow Cytometry, Western Blot, Expressing, Fluorescence, Derivative Assay

Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Western blot and immunofluorescence staining showing the expression of relevant cytokines in the CIB of 2VO + EMS rats. (A) Representative images of triple immunofluorescence staining showing Tie2 + , CD11b + , and CD206 + cells in the CIB of each group. Bar = 50 μm. (B) Counts of M2 TEMs (Tie2 + /CD11b + /CD206 + ) in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (C) Representative western blot showing the relative expression of ANGPT2, CD206, and ARG1 (normalized to β‐Actin expression). (D) Densitometric analyses of the relative expression of ANGPT2, CD206, and ARG1 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (E) Representative images of double immunofluorescence staining showing CD31 + and Ki67 + cells in the CIB of each group. Bar = 50 μm. (F) Column chart showing the counts of CD31 + cells in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (G) Column chart showing the proportion of Ki67‐positive ECs in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (H) Representative western blot showing the relative expression of CD31 (normalized to β‐Actin expression). (I) Densitometric analyses of the relative expression of CD31 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (H) * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The error bars represent the ±SDs. CIB: Chronically ischaemic brain; TEMs, Tie2‐expressing monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Western Blot, Immunofluorescence, Staining, Expressing, Double Immunofluorescence Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: ANGPT2 /Tie2 Enhances H3K18la ‐Mediated Macrophage M2 Polarization to Promote Endothelial Cell Proliferation in the Chronically Ischaemic Brain

doi: 10.1002/cns.70879

Figure Lengend Snippet: Behavioral test, western blotting, and immunofluorescence results in 2VO + EMS rats 4‐week post‐modeling. (A) Quantitative analysis of the percentage of recognition preference for the novel object in each group rats in NORT test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (B) Representative image showing the swimming paths in each group rats during the MWM test. (C) Line chart showing the average escape latencies in each group rats during the MWM test ( n = 6; two‐way ANOVA and Tukey's multiple comparisons test; ***: TEMs vs. TEMs ANGPT2 , ****: TEMs ANGPT2 vs. TEMs ANGPT2+Oxamate , TEMs ANGPT2 vs. TEMs ANGPT2+C646 , TEMs ANGPT2 vs. TEMs ANGPT2+LPS ). (D) Quantitative analysis of the time spent in target quadrant in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (E) Quantitative analysis of the number of platform crossings in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (F) Quantitative analysis of the average swimming speed in each group rats during the MWM test ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (G) Representative western blot showing the relative expression of CD31 (normalized to β‐Actin expression). (H) Densitometric analyses of the relative expression of CD31 ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (I) Representative images of double immunofluorescence staining showing CD31 + and Ki67 + cells in the CIB of each group. Bar = 50 μm. (J) Column chart showing the counts of CD31 + cells in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). (K) Column chart showing the proportion of Ki67‐positive ECs in each group ( n = 6; one‐way ANOVA and Tukey's multiple comparisons test). The error bars represent the ± SDs. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. 2VO, 2‐vessel occlusion; EMS, encephalomyosynangiosis; MWM, Morris water maze; NORT, novel object recognition test; TEMs, Tie2‐expressing monocytes/macrophages.

Article Snippet: In accordance with previous studies, primary TEMs and TNMs were treated with 100 ng/mL ANGPT2 protein (HY‐P7510; MedChemExpress, USA) for 3 h, while TDMs were exposed to the same concentration of ANGPT2 for 6 h.

Techniques: Western Blot, Immunofluorescence, Expressing, Double Immunofluorescence Staining

Journal: Cell Death & Disease

Article Title: Decreased ZNF750 promotes angiogenesis in a paracrine manner via activating DANCR/miR-4707-3p/FOXC2 axis in esophageal squamous cell carcinoma

doi: 10.1038/s41419-020-2492-2

Figure Lengend Snippet: a PCR array in ZNF750 knockdown cells using the kit of Cancer Pathway Finder PCR Array. b , c qRT-PCR showed the expression of FOXC2 in ZNF750 knockdown cells ( b ) and ZNF750-wt cells ( c ). GAPDH was performed as a loading control. Statistical analysis was performed with a two-sided t test. d Representative images of tube formation ( upper ), migration ( middle ) and invasion( lower ) of HUVEC. e The conditioned medium from FOXC2 knockdown in ZNF750 knockdown cells reversed the effect on tube formation ( left ), migration ( middle ) and invasion( right ) of HUVEC. f Angiogenic biomarkers ANGPT2 , FLT1 , CCL2 and FGF2 were detected by qRT-PCR in ZNF750 knockdown cells and FOXC2 knockdown in ZNF750 knockdown cells. Statistical analysis was performed with one-way ANOVA. ** p < 0.01, *** p < 0.0001.

Article Snippet: The antibodies used in this experiment are shown as follows: Ki-67 (Zhongshan, China, ZM-0166), ZNF750 (Sigma, USA, HPA021573), FOXC2 (Proteintech, USA, 23066-1-AP), CD31(Abcam, USA, ab134168), FLT1 (Proteintech, USA, 13687-1-AP), ANGPT2 (Proteintech, USA, 24613-1-AP).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, Control, Migration

Journal: World journal of surgical oncology

Article Title: Homeobox D9 drives the malignant phenotypes and enhances the Programmed death ligand-1 expression in non-small cell lung cancer cells via binding to Angiopoietin-2 promoter.

doi: 10.1186/s12957-023-02969-z

Figure Lengend Snippet: Fig. 4 ANGPT2 is a direct transcriptional target of HOXD9. A Correlation analysis of HOXD9 with ANGPT2 in NSCLC using the UALCAN and TIMER databases. B NCI-H661 cells were transfected with exHOXD9 or empty vector, while NCI-H292 cells were transfected with siHOXD9#1, siHOXD9#2 or their negative control siNC. After 48 h of transfection, ANGPT2 mRNA expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by real time-PCR assay. C After 48 h of transfection, ANGPT2 protein expression in two NSCLC cells was evaluated by western blot assay. D HEK293T cells were co-transfected with a pGL3-basic vector containing the promoter sequence of homo ANGPT2, pRL-TK, and exHOXD9 or empty vector. After 24 h of transfection, ANGPT2 luciferase activity was evaluated by dual-luciferase reporter assay. * p < 0.05; *** p < 0.001. Data in (B) and (D) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials

Article Snippet: After blocking with 5% (m/v) skim milk (Cat No., A600669, Sangon Biotech, Shanghai, China), the membrane was probed with primary antibodies against HOXD9 (1:1000 dilution, rabbit polyclonal; Cat No., 20,560–1-AP, ProteinTech, Wuhan, China), cleaved poly (ADP-ribose) polymerase 1 (PARP1; 1:500 dilution, rabbit monoclonal; Cat No., 5625, CST, Danvers, MA, USA), cleaved caspase-3 (1:1000 dilution, rabbit monoclonal; Cat No., 9661, CST), cyclin E (1:500 dilution, rabbit polyclonal; Cat No., AF0144, Affinity Biosciences, Changzhou, China), cyclin B1 (1:1000 dilution, rabbit monoclonal; Cat No., A19037, ABclonal, Wuhan, China), ANGPT2 (1:1000 dilution, rabbit monoclonal; Cat No., A11306, ABclonal), PD-L1 (1:1000 dilution, rabbit polyclonal; Cat No., DF6526, Affinity Biosciences) and GAPDH (1:10,000 dilution, mouse monoclonal; Cat No., 60,004–1-Ig, ProteinTech) at 4 °C overnight.

Techniques: Transfection, Plasmid Preparation, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Sequencing, Luciferase, Activity Assay, Reporter Assay

Journal: World journal of surgical oncology

Article Title: Homeobox D9 drives the malignant phenotypes and enhances the Programmed death ligand-1 expression in non-small cell lung cancer cells via binding to Angiopoietin-2 promoter.

doi: 10.1186/s12957-023-02969-z

Figure Lengend Snippet: Fig. 5 ANGPT2 is required for HOXD9-mediated proliferation, cell cycle progress and apoptotic inhibition of NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and the plasmid expressing ANGPT2 (exANGPT2) or empty vector. NCI-H661 cells were co-transfected with exHOXD9 and siANGPT2 or siNC. A After 48 h of transfection, ANGPT2 mRNA expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by real time-PCR assay. B After 48 h of transfection, ANGPT2 protein expression in two NSCLC cells (NCI-H661 and NCI-H292) was evaluated by western blot assay. C After 48 h of transfection, NSCLC cells were seeded into 96-well microplates and analyzed with CCK-8 reagents at indicated time. D Cell cycle progression in two NSCLC cells was evaluated by flow cytometry analysis. E Apoptosis in NCI-H292 cells was evaluated by flow cytometry analysis. The apoptotic rate was quantified by adding the percentages of early apoptotic cells (Annexin V+/PI− in Q4 quadrant) and late apoptotic cells (Annexin V+/PI+ in Q2 quadrant). * p < 0.05; ** p < 0.01; *** p < 0.001. Data in (A), (C), (D) and (E) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials

Article Snippet: After blocking with 5% (m/v) skim milk (Cat No., A600669, Sangon Biotech, Shanghai, China), the membrane was probed with primary antibodies against HOXD9 (1:1000 dilution, rabbit polyclonal; Cat No., 20,560–1-AP, ProteinTech, Wuhan, China), cleaved poly (ADP-ribose) polymerase 1 (PARP1; 1:500 dilution, rabbit monoclonal; Cat No., 5625, CST, Danvers, MA, USA), cleaved caspase-3 (1:1000 dilution, rabbit monoclonal; Cat No., 9661, CST), cyclin E (1:500 dilution, rabbit polyclonal; Cat No., AF0144, Affinity Biosciences, Changzhou, China), cyclin B1 (1:1000 dilution, rabbit monoclonal; Cat No., A19037, ABclonal, Wuhan, China), ANGPT2 (1:1000 dilution, rabbit monoclonal; Cat No., A11306, ABclonal), PD-L1 (1:1000 dilution, rabbit polyclonal; Cat No., DF6526, Affinity Biosciences) and GAPDH (1:10,000 dilution, mouse monoclonal; Cat No., 60,004–1-Ig, ProteinTech) at 4 °C overnight.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: World journal of surgical oncology

Article Title: Homeobox D9 drives the malignant phenotypes and enhances the Programmed death ligand-1 expression in non-small cell lung cancer cells via binding to Angiopoietin-2 promoter.

doi: 10.1186/s12957-023-02969-z

Figure Lengend Snippet: Fig. 6 ANGPT2 is involved in the anti-migratory and anti-invasive effects of HOXD9 silencing in NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and exANGPT2 or empty vector. NCI-H661 cells were co-transfected with exHOXD9 and siANGPT2 or siNC. (A and B) After 48 h of transfection, the migratory ability and invasiveness of NCI-H292 (A) and NCI-H661 (B) cells were evaluated by scratch-wound healing assay (Scale bar = 200 μm) and transwell assay (Scale bar = 100 μm). ** p < 0.01; *** p < 0.001. Data in (A) and (B) are presented as mean values ± SD

Article Snippet: After blocking with 5% (m/v) skim milk (Cat No., A600669, Sangon Biotech, Shanghai, China), the membrane was probed with primary antibodies against HOXD9 (1:1000 dilution, rabbit polyclonal; Cat No., 20,560–1-AP, ProteinTech, Wuhan, China), cleaved poly (ADP-ribose) polymerase 1 (PARP1; 1:500 dilution, rabbit monoclonal; Cat No., 5625, CST, Danvers, MA, USA), cleaved caspase-3 (1:1000 dilution, rabbit monoclonal; Cat No., 9661, CST), cyclin E (1:500 dilution, rabbit polyclonal; Cat No., AF0144, Affinity Biosciences, Changzhou, China), cyclin B1 (1:1000 dilution, rabbit monoclonal; Cat No., A19037, ABclonal, Wuhan, China), ANGPT2 (1:1000 dilution, rabbit monoclonal; Cat No., A11306, ABclonal), PD-L1 (1:1000 dilution, rabbit polyclonal; Cat No., DF6526, Affinity Biosciences) and GAPDH (1:10,000 dilution, mouse monoclonal; Cat No., 60,004–1-Ig, ProteinTech) at 4 °C overnight.

Techniques: Transfection, Plasmid Preparation, Wound Healing Assay, Transwell Assay

Journal: World journal of surgical oncology

Article Title: Homeobox D9 drives the malignant phenotypes and enhances the Programmed death ligand-1 expression in non-small cell lung cancer cells via binding to Angiopoietin-2 promoter.

doi: 10.1186/s12957-023-02969-z

Figure Lengend Snippet: Fig. 8 ANGPT2 blocks the suppression of PD-L1 expression by HOXD9 in NSCLC cells. NCI-H292 cells were co-transfected with siHOXD9 and exANGPT2 or empty vector. A After 48 h of transfection, total PD-L1 protein expression in NCI-H292 cells was evaluated by western blot assay. B After 48 h of transfection, membrane PD-L1 protein expression in NCI-H292 cells was evaluated by flow cytometry analysis. MFI, relative mean fluorescence intensity. * p < 0.05. Data in (B) are presented as mean values ± SD. The blots were cropped and the original uncropped images of blots were shown in supplementary materials

Article Snippet: After blocking with 5% (m/v) skim milk (Cat No., A600669, Sangon Biotech, Shanghai, China), the membrane was probed with primary antibodies against HOXD9 (1:1000 dilution, rabbit polyclonal; Cat No., 20,560–1-AP, ProteinTech, Wuhan, China), cleaved poly (ADP-ribose) polymerase 1 (PARP1; 1:500 dilution, rabbit monoclonal; Cat No., 5625, CST, Danvers, MA, USA), cleaved caspase-3 (1:1000 dilution, rabbit monoclonal; Cat No., 9661, CST), cyclin E (1:500 dilution, rabbit polyclonal; Cat No., AF0144, Affinity Biosciences, Changzhou, China), cyclin B1 (1:1000 dilution, rabbit monoclonal; Cat No., A19037, ABclonal, Wuhan, China), ANGPT2 (1:1000 dilution, rabbit monoclonal; Cat No., A11306, ABclonal), PD-L1 (1:1000 dilution, rabbit polyclonal; Cat No., DF6526, Affinity Biosciences) and GAPDH (1:10,000 dilution, mouse monoclonal; Cat No., 60,004–1-Ig, ProteinTech) at 4 °C overnight.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Membrane, Flow Cytometry, Fluorescence