Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 1 | High ACE2 expression was linked to increased VM and better prognosis in NSCLC. (A) Typical image of ACE2, VE-cadherin, EphA2 protein expression and CD34/PAS double staining in TMA tissues. Case B1 had massive CD34−/PAS+ VM (yellow arrows) lined by ACE2, VE-cadherin and EphA2 high expressing tumor cells. Case G1 had abundant CD34+/PAS−MVs (black arrows) with ACE2, VE-cadherin and EphA2 low expressing tumor cells. (B) Kaplan-Meier analysis of OS in NSCLC patients with ACE2 low or high expression. P = 0.044.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Double Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 2 | ACE2-induced better outcome in NSCLC patients might be attributed to less vessels and more VM formation. (A, C, E, G) Typical tissue images of each group stained with ACE2 or CD34/PAS. Yellow arrows: CD34−/PAS+ VMs; black arrows: CD34+/PAS−MV. (B, D, F, H) Kaplan–Meier analysis of OS in each group.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 3 | Human ACE2 was stably overexpressed in A549-ACE2-OE cells. (A) Schematic representation of pLenti6.3-MCS/V5 DEST. (B) pLenti6.3-ACE2 expression vector was detected by PCR. (C) Fluorescence of EGFP in A549-ACE2-OE cells (left) and parental cells (right) was determined by fluorescence microscopy. (D) RT-PCR experiment of ACE2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, *p < 0.05. (E) Western blot analysis of ACE2 expression level in A549-ACE2-OE cells and parental cells. (F) Quantification of ACE2 expression level in A549-ACE2-OE cells and parental cells. Mean ± SD, n = 3, ***p < 0.001.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Fluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 4 | Tube formation ability of A549 cells was improved with ACE inhibitory state. (A) Morphologies of a panel of A549-ACE2-OE cells, A549-NC cells, and A549-NC cells treated with ACEI (1, 5, and 10 nM/L) were shown as sheet-like and thread-like cell types in 2D culture, which were outlined partly with yellow lines. Representative images were shown above. Upper: white light; lower: fluorescence. (B) Quantification of sheet-like or thread-like cells and pebble-like cells in three groups, representatively, Mean ± SD, n = 3, **p < 0.01. (C) Images of both above cell lines grown in 3D Matrix gel. A 10 nM/L ACEI dilution was performed. Representative images were shown above. Yellow arrows point out the free cancer cells escaping from tube wall and isolated segments. Upper: white light; lower: fluorescence. (D, E) Images of 3D culture were applied to determine average number of nodes, branches, isolated segments, meshes and mean mesh area in those groups, per field, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. ns, no significance.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Isolation

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 5 | VM formation was increased, and vasculature was lessened due to inhibition of RAS in vivo. (A) Growth curve of allograft tumors of A549-ACE2-OE cells, A549-NC cells with or without ACEI treatment, Mean ± SD, n = 3, *p < 0.05. ns, no significance. (B) Weight of resected tumors, Mean ± SD, n = 3, **p < 0.01, *p < 0.05. (C) Continuous sections of allograft tumor tissues stained with PAS, CD34, VE-cadherin, or EphA2 immunohistochemical stain. Black arrow points out a typical MV (CD34+/PAS−); yellow arrows point out typical VM (CD34−/PAS+). (D) Quantification of MV and VM in different groups, Mean ± SD, n = 3, per field, ***p < 0.001, **p < 0.01. (E) Quantification of VE-cadherin and EphA2 mean optical density in three groups, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Inhibition, In Vivo, Staining, Immunohistochemical staining

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 6 | VE-cadherin and EphA2 expression was upregulated in A549 cells and NSCLC tissues with impaired local RAS status. (A, B) RT-PCR experiment of VE-cadherin and EphA2 mRNA level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (C–E) Western blot analysis and quantification of VE- cadherin and EphA2 expression level in A549-ACE2-OE cells and control cells, Mean ± SD, n = 3, ***p < 0.001. (F, G) Linear regressions of VM number and VE- cadherin (P < 0.0001) or EphA2 (P = 0.0108) score in TMA. (H) Typical tissue images of both groups stained with VE-cadherin, ACE2 or CD34/PAS. Case F13 with ACE2 low status was provided with rambling VM covered by tumor cells which only expressed VE-cadherin in nuclei; case D6 with ACE2 high status had ordered VM lined by tumor cells expressing VE-cadherin in both nuclei and cytomembranes. Red arrow: VE-cadherin membrane expression.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Staining, Membrane

Journal: Frontiers in oncology

Article Title: The Angiotensin-Converting Enzyme Inhibitory State Promotes the Transformation of Non-Small Cell Lung Cancer Blood Supply Pattern Toward Vasculogenic Mimicry Formation.

doi: 10.3389/fonc.2021.663671

Figure Lengend Snippet: FIGURE 7 | PI3K/AKT, p38MAPK, and HIF1-a were inactivated, and Nodal/Notch4 pathway was activated in A549-ACE2-OE cell model. (A–E) Western blot analysis and quantification of AKT, p-AKT, p38, and p-p38 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01. (F, G) Immunoflurescence assay and quantification of HIF1-a mean optical density in A549-ACE2-OE cells and negative control, Mean ± SD, n = 4, *p < 0.05. (H, I) Western blot analysis and quantification of Nodal and Notch4 expression level in A549-ACE2-OE cells and negative control, Mean ± SD, n = 3, ***p < 0.001, **p < 0.01.

Article Snippet: The sections were dewaxed, rehydrated, treated with antigen retrieval, hatched with primary antibodies of ACE2 (1:200), VE-cadherin (1:200), EphA2 (1:100), and CD34 (1:200, ab81289, Abcam) overnight at 4°C, individually exposed to secondary antibody (SA1020, BOSTER, USA) 1 h at RT, and colored with DAB peroxidase substrate.

Techniques: Western Blot, Expressing, Negative Control

Journal: Environmental science and pollution research international

Article Title: Rutin protects against gamma-irradiation and malathion-induced oxidative stress and inflammation through regulation of mir-129-3p, mir-200C-3p, and mir-210 gene expressions in rats' kidney.

doi: 10.1007/s11356-023-27166-z

Figure Lengend Snippet: Fig. 7 The activity of acetylcholinesterase, angiotensin I converting enzyme, and angiotensin II converting enzyme in the kidney tissues. C, control; rutin, rutin-treated animals; IRR, gamma-irradiated ani- mals; MT, malathion-treated animals; IRR/MT, gamma-irradiation/ malathion-treated animals; IRR/rutin, gamma-irradiation/rutin-treated animals; MT/rutin, malathion/rutin-treated animals; IRR/MT/rutin, gamma-irradiation/malathion/rutin-treated animals; AchE, acetylcho- linesterase; ACE I, angiotensin I converting enzyme. The statistical significance to control, IRR, MT, and IRR/MT are denoted by a, b, c, and d, respectively, at p < 0.05. Statistical significance was analyzed by one-way ANOVA with Tukey post hoc multiple comparisons. These enzymes are assessed by ELISA technique

Article Snippet: Elabscience ELISA commercial kits for rats were used to assess the activity of angiotensin-converting enzymes ACE I (Cat. No: E-EL-R2401) and ACE II (Cat. No: E-ELR2453), in the rats’ kidneys tissues, according to the catalogs’ instructions.

Techniques: Activity Assay, Control, Irradiation, Enzyme-linked Immunosorbent Assay

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: EEF1AKMT4 trimethylates eEF1A2 at K36 site in GBC. (A) Spatial distribution of the five main methylation sites in the protein structure of eEF1A2 combined with GDP (PDB: 6ra9). Most of the methylation sites were located in the nucleotide-binding domain of eEF1A2. (B) Relative mRNA expression of five methylases, EEF1AKMT4, METTL13, N6AMT2, METTL21B, and METTL10, which are responsible for methylation of K36, K55, K79, K165, and K318 sites, respectively, was quantified by qRT-PCR in 10 cases of gallbladder cancer tissues and their patient-paired normal tissues. (C) Coomassie blue staining of anti-eEF1A2 co-immunoprecipitation in GBCSD, SGC996, and HEK293T cell lines. Target bands are indicated by a black box and were cut and subjected to LC-MS/MS to analyze the methylation status. (D) Methylation status at the K36, K55, K79, K165, and K318 sites of eEF1A2 in GBCSD, SGC996, and HEK293T cell lines were analyzed. (E) Representative tandem mass spectra identifying in vitro tri-methylated (upper) and non-methylated (lower) eEF1A2K36. m/z for b and y ions observed in the spectra are indicated in red and blue, respectively. (F) Histogram showing the methylation changes in K36, K55, K79, K165, and K318 before (upper) and after (lower) knockdown of EEF1AKMT4 in GBCSD cells. (G) Selected ion chromatograms for non-, mono-, di-, and trimethyl eEF1AK36 peptides from GluC digestion of endogenous eEF1A2 immunoprecipitated from whole-cell lysates of GBCSD, indicating the methylation status shift after EEF1AKMT4 knockdown. (H) Western blot analysis of the knockdown efficiency of EEF1AKMT4 in GBCSD and the status of eEF1A2 and eEF1A2K36me3. (I) Expression of eEF1AKMT4 and eEF1A2 K36me3 levels in GBC tumor tissues and paired normal tissues were examined by Western blotting and analyzed using ImageJ. Statistical significance between groups was assessed using Student's t-test. ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Methylation, Binding Assay, Expressing, Quantitative RT-PCR, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, In Vitro, Knockdown, Western Blot

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: Knockdown of EEF1AKMT4 inhibits the malignant phenotype of GBC while its overexpression is not tumor-promoting. (A–B) Expression levels of EEF1AKMT4 in HEK293T, RBE, NOZ, GBCSD, SGC996, and OCUG-1 were detected using qRT-PCR (A) and Western blotting (B). (C) EEF1AKMT4 was knocked down or overexpressed in the GBCSD and SGC996 cell lines, and validated by Western blotting. (D) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 knockdown in GBCSD and SGC996 cells. (E) Clone formation assay was performed in EEF1AKMT4 knockdown and vector control cells in GBCSD and SGC996 cells. (F) Wound healing assays were performed to investigate the effect of EEF1AKMT4 knockdown on the migration ability of the GBCSD and SGC996 cell lines. (G) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 knockdown. (H) CCK-8 assays were performed to identify alterations in cell proliferation after EEF1AKMT4 overexpression in GBCSD and SGC996 cells. ( I ) Transwell assays with matrigel were performed to evaluate the invasive ability of GBCSD and SGC996 cells after EEF1AKMT4 overexpression. Statistical significance between subgroups was assessed using the Student's t-test. ns, not significant; ∗∗∗, p < 0.001.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Knockdown, Over Expression, Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Tube Formation Assay, Plasmid Preparation, Control, Migration

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: K36 site trimethylation is essential for the tumor-promoting effect of eEF1A2. (A) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (B) CCK-8 assays in eEF1A2-knockdown GBCSD SGC996 cells complemented with vector, eEF1A2 WT, or eEF1A2 K36R. (C) Clone formation assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (D) Wound healing assays of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with vector, eEF1A2 WT or eEF1A2 K36R. (E) Transwell assays with matrigel of cells treated with scrambled shRNA, or eEF1A2-knockdowned GBCSD SGC996 cells complemented with the vector, eEF1A2 WT, or eEF1A2 K36R. (F) Western blot analysis of the expression of EEF1AKMT4, eEF1A2 K36me3, and eEF1A2 in EEF1AKMT4-knockdown GBCSD cells complemented with vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (G) CCK-8 assays in EEF1AKMT4-knockdown GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (H) Clone formation assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (I) Wound healing assays of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with vector, EEF1AKMT4 WT or EEF1AKMT4 D88A. (J) Transwell assays with matrigel of cells treated with scrambled shRNA, or EEF1AKMT4-knockdowned GBCSD SGC996 cells complemented with the vector, EEF1AKMT4 WT, or EEF1AKMT4 D88A. (K) In vivo subcutaneous xenografts were established. (L–M) Tumor weight (L) and volume (M) of the subcutaneous xenografts were measured. (N) Comparison of popliteal lymph node size. (O) Comparison of the volume of metastasized popliteal lymph nodes. (P) Representative images of a nude mouse model of popliteal LN metastasis. HE staining of the lymph nodes was performed. Scale bar: 100 μm ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Western Blot, Expressing, Knockdown, Plasmid Preparation, CCK-8 Assay, shRNA, In Vivo, Comparison, Staining

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: eEF1A2 K36 trimethylation affects protein output by affecting its GTP enzyme activity. (A) FLAG-tagged eEF1A2 WT and eEF1A2 K36R were overexpressed in HEK293T cells. Flag-co-IP and subsequent LC-MS/MS were performed to identify the differences in binding proteins between eEF1A2 WT and K36R mutations. The binding proteins of eEF1A2 WT (left) and eEF1A2 K36R (right) in HEK293T cells were analyzed by KEGG enrichment analysis. (B) Heatmap showing the differentially expressed genes after eEF1A2 knockdown in GBCSD cell lines. (C) The differentially expressed genes after eEF1A2 knockdown were analyzed by KEGG enrichment analysis. (D) Western blot analysis of the expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (E) qPCR analysis of the mRNA expression of ERK1/2 and AKT in EEF1AKMT4-knockdown or eEF1A2-knockdown GBCSD cells. (F) SUnSET assays were performed under the indicated conditions to analyze the effect of eEF1A2 K36 methylation status on the protein synthesis rate in GBCSD (left) and SGC996(right) cell lines. These results revealed reduced protein production in eEF1A2 K36me0 cells. (G) Purification of eEF1A2 ± K36me3 protein with anti-eEF1A2 co-immunoprecipitation in EEF1AKMT wt and knockdown cells. Top panel: Western blot validation of EEF1AKMT4; middle panels: Western blot analysis with the indicated antibodies against eEF1A2 purified from 293 T cells; bottom panel: Coomassie stain of purified eEF1A2 protein. (H) In vitro GTP hydrolysis by trimethylated or unmethylated eEF1A2. eEF1A2 ± K36me3 purified was incubated with increasing amounts of GTP at 37 °C for 3 h. Kinetic parameters were obtained by fitting the Michaelis–Menten equation to plot the velocity of phosphate formation against GTP concentration. (I) K36me3 increases the catalytic efficiency of GTP hydrolysis by eEF1A2. The Michaelis–Menten kinetic parameters of eEF1A2 ± K36me3 are shown.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, Liquid Chromatography with Mass Spectroscopy, Binding Assay, Knockdown, Western Blot, Expressing, Methylation, Purification, Immunoprecipitation, Biomarker Discovery, Staining, In Vitro, Incubation, Concentration Assay

Journal: Genes & Diseases

Article Title: EEF1AKMT4-eEF1A2 synergistically facilitates the progression of GBC by promoting ribosomal protein output

doi: 10.1016/j.gendis.2025.101619

Figure Lengend Snippet: A schematic model shows the effects of EEF1AKMT4 and eEF1A2 on regulating ribosomal protein synthesis. EEF1AKMT4 trimethylates eEF1A2 at K36 and fuels its GTPase activity. Elevated eEF1A2 enzyme activity promotes the protein output of several oncogenic growth signals including AKT and ERK. Overall, upregulated eEF1A2 expression together with the fuel of EEF1AKMT4 promotes the progression of GBC and LN metastasis.

Article Snippet: EEF1AKMT4 , Proteintech , Cat No. 15418-1-AP.

Techniques: Activity Assay, Expressing