Journal: bioRxiv

Article Title: Angiotensin II- Angiotensin II Receptor Type 1 Signaling Facilitates Gastric Cancer Metastasis via Kruppel-like Factor 4 Suppression and Tight Junction Breakdown

doi: 10.64898/2026.01.08.698396

Figure Lengend Snippet: (A) Representative IHC staining shows that the expression of ATII and AT1R is significantly elevated in GC tissues compared with adjacent normal gastric tissues. Original magnification, x20 (B) The violin plot shows that the IHC score for ATII and AT1R is significantly higher in non-metastatic and metastatic GC tissues than in adjacent normal gastric tissues. Significance was determined by 1-way ANOVA ( n = 64). ( C ) TCGA data show the expression of AGT and a stage-wise increase in expression of AGT in STAD. ( D) Kaplan-Meier survival curves from TCGA show OS and DFS of STAD patients grouped by AGT and AGTR1 expressions. High AGT expression is associated with significantly poor OS ( p = 0.02), and high AGTR1 expression is associated with significantly poor DFS ( p = 0.0035). GC, gastric cancer; IHC, immunohistochemistry; STAD, stomach adenocarcinoma; OS, overall survival; DFS, disease-free survival; TCGA, the cancer genome atlas; AGT, Angiotensin II; AGTR1, Angiotensin II receptor 1 . **P < 0.01 ; ****P < 0.0001. ns: no significance.

Article Snippet: The antibodies ATII (Cat# NB100-62346 Novus Biologicals, CO), AT1R (Cat# ab124505 Abcam, MA), KLF4 (Cat# ab216968 Abcam, MA and Cat# 4038S Cell Signaling Technology, Danvers, MA), claudin 1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), claudin 3 (Cat# NBP2-46299 Novus Biologicals, CO), claudin 4 (Cat# ab53156, Abcam, MA), Zonula occludens 1 (Cat# NBP2-80141 Novus Biologicals, CO), GAPDH (Cat#2118 Cell Signaling Technology, MA), HDAC1 (Cat# NB100-56340 Novus Biologicals, CO) were used for western blotting ( ).

Techniques: Immunohistochemistry, Expressing

Journal: bioRxiv

Article Title: Angiotensin II- Angiotensin II Receptor Type 1 Signaling Facilitates Gastric Cancer Metastasis via Kruppel-like Factor 4 Suppression and Tight Junction Breakdown

doi: 10.64898/2026.01.08.698396

Figure Lengend Snippet: (A) Enzymatic Immuno Assay (EIA), western blot, and qRT-PCR reveal expression of ATII in GC cells, MKN45, MKN7, AGS, and NCI-N87. For quantification, the mRNA level of the ATII gene was normalized to the housekeeping gene GAPDH. Data represented as mean±SD of three independent experiments. (B) Western blot and qRT-PCR data show the expression of AT1R in GC cells. For quantification, the mRNA level of the ATIR gene was normalized to the housekeeping gene GAPDH. Data represented as mean±SD of three independent experiments. Blockade of AT1R by the AT1R antagonist losartan (1µM) significantly reduces GC cell (C) proliferation, (D) migration (Original magnification, ×4), and (E) invasion (Original magnification, ×20). For the proliferation the data represented as mean±SEM and analyzed using 2-tailed t test ( n = 5). The percentage of wound closure is shown in bar diagrams. Data represented as mean±SD and analyzed using 2-tailed t test ( n = 3). Transwell assay shows that losartan treatment significantly reduces the invasive ability of MKN45 and NCI-N87 GC cells by inhibition of the AT1R receptors expressed in these cells. The number of invaded cells through the membrane in the control and losartan-treated groups is represented as a bar diagram. Data represented as mean±SEM and analyzed using 2-tailed t test ( n = 9). ( F) A volcano plot illustrating the differentially expressed genes (DEGs) obtained from RNA-seq analysis of losartan-treated MKN45 GC cancer cells compared to control cells. Losartan-treated GC cells with upregulated and downregulated genes are shown in red and blue colors respectively. ( G) The heatmap illustrates the differential expression of TJ-related genes in losartan-treated MKN45 cells compared to control cells. ( H) GSEA plot demonstrates a positive enrichment of the KEGG TJ gene set in losartan-treated MKN45 cells, suggesting that TJs are more intact upon treatment with losartan. I , Violin plot depicts the overall expression of TJ-related genes in losartan-treated MKN45 cells compared with control cells. *** P < 0.001, **** P < 0.0001. GC, gastric cancer; EIA, enzymatic immunoassay; GSEA, gene set enrichment analysis; KEGG, Kyoto encyclopedia of genes and genomes.

Article Snippet: The antibodies ATII (Cat# NB100-62346 Novus Biologicals, CO), AT1R (Cat# ab124505 Abcam, MA), KLF4 (Cat# ab216968 Abcam, MA and Cat# 4038S Cell Signaling Technology, Danvers, MA), claudin 1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), claudin 3 (Cat# NBP2-46299 Novus Biologicals, CO), claudin 4 (Cat# ab53156, Abcam, MA), Zonula occludens 1 (Cat# NBP2-80141 Novus Biologicals, CO), GAPDH (Cat#2118 Cell Signaling Technology, MA), HDAC1 (Cat# NB100-56340 Novus Biologicals, CO) were used for western blotting ( ).

Techniques: Immuno Assay, Western Blot, Quantitative RT-PCR, Expressing, Migration, Transwell Assay, Inhibition, Membrane, Control, RNA Sequencing, Quantitative Proteomics, Enzyme Immunoassay

Journal: bioRxiv

Article Title: Angiotensin II- Angiotensin II Receptor Type 1 Signaling Facilitates Gastric Cancer Metastasis via Kruppel-like Factor 4 Suppression and Tight Junction Breakdown

doi: 10.64898/2026.01.08.698396

Figure Lengend Snippet: (A) IHC staining shows that the expression of TJ proteins Claudin 1, 3, 4, and Zonula occludens 1 is markedly decreased in human GC tissues compared to adjacent normal gastric tissues. The IHC score was analyzed and shown by the dot plot comparing IHC scores for Claudin 1 ( n = 26 for NS; n = 27 for GC), 3 ( n = 19 for NS; n = 19 for GC), 4 ( n = 21 for NS; n = 19 for GC), and Zonula occludens 1 ( n = 22 for NS; n = 21 for GC) in two groups: NS and GC. Original magnification, ×20. The IHC score for each TJ protein is significantly higher in normal stomach tissues compared to GC tissues. Each dot represents an individual data point, and the horizontal lines represent the median for each group. Data analyzed using 2-tailed t test. ( B) Expression of ATII in human GC tissues shows an inverse correlation with the expression of TJ proteins, Claudin 1 ( n = 40), 3 ( n = 40), 4 ( n = 43), and Zonula occludens 1 ( n = 44). Correlation analysis between ATII expression and Claudins was performed using Pearson’s correlation test and the values are displayed on the graph. ( C) Expression of AT1R in human GC tissues shows an inverse correlation with the expression of TJ proteins, Claudin 1 ( n = 40), 3 ( n = 40), 4 ( n = 43), and Zonula occludens 1 ( n = 44). Correlation analysis between ATIR expression and Claudins was performed using Pearson’s correlation test and the values are displayed on the graph. ( D) IHC images and quantification of KLF4-positive cells indicate a gradual loss of KLF4 expression in GC tissues with disease progression. Original magnification, ×20. Significance was determined by 1-way ANOVA ( n = 64). (E) Quantitative analysis reveals a strong positive correlation between the percentage of KLF4-positive cells and the expression of these TJ proteins, Claudin 1 ( n = 40), 3 ( n = 40), 4 ( n = 43), and Zonula occludens 1 ( n = 43) in GC tissues. Correlation analysis between KLF4 expression and Claudins was performed using Pearson’s correlation test and the values are displayed on the graph. ** P < 0.01;*** P < 0.001; **** P < 0.0001. ns : no significance. GC, gastric cancer; TJ, tight junction; NS, normal stomach; IHC, immunohistochemistry.

Article Snippet: The antibodies ATII (Cat# NB100-62346 Novus Biologicals, CO), AT1R (Cat# ab124505 Abcam, MA), KLF4 (Cat# ab216968 Abcam, MA and Cat# 4038S Cell Signaling Technology, Danvers, MA), claudin 1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), claudin 3 (Cat# NBP2-46299 Novus Biologicals, CO), claudin 4 (Cat# ab53156, Abcam, MA), Zonula occludens 1 (Cat# NBP2-80141 Novus Biologicals, CO), GAPDH (Cat#2118 Cell Signaling Technology, MA), HDAC1 (Cat# NB100-56340 Novus Biologicals, CO) were used for western blotting ( ).

Techniques: Immunohistochemistry, Expressing, Biomarker Discovery

Journal: bioRxiv

Article Title: Angiotensin II- Angiotensin II Receptor Type 1 Signaling Facilitates Gastric Cancer Metastasis via Kruppel-like Factor 4 Suppression and Tight Junction Breakdown

doi: 10.64898/2026.01.08.698396

Figure Lengend Snippet: (A) The quantitative correlation analysis shows a significant negative correlation between the average IHC score for ATII/AT1R expression and the number of KLF4-positive cells. Correlation was performed using Pearson’s correlation test and the values are displayed on the graph ( n = 46). (B) cBioPortal data show that elevated mRNA expression of AGT and AGTR1 is negatively correlated with KLF4 expression in GC. *P < 0.05 ; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001. GC, gastric cancer; TJ, tight junction.

Article Snippet: The antibodies ATII (Cat# NB100-62346 Novus Biologicals, CO), AT1R (Cat# ab124505 Abcam, MA), KLF4 (Cat# ab216968 Abcam, MA and Cat# 4038S Cell Signaling Technology, Danvers, MA), claudin 1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), claudin 3 (Cat# NBP2-46299 Novus Biologicals, CO), claudin 4 (Cat# ab53156, Abcam, MA), Zonula occludens 1 (Cat# NBP2-80141 Novus Biologicals, CO), GAPDH (Cat#2118 Cell Signaling Technology, MA), HDAC1 (Cat# NB100-56340 Novus Biologicals, CO) were used for western blotting ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: Angiotensin II- Angiotensin II Receptor Type 1 Signaling Facilitates Gastric Cancer Metastasis via Kruppel-like Factor 4 Suppression and Tight Junction Breakdown

doi: 10.64898/2026.01.08.698396

Figure Lengend Snippet: (A) Schematic representation of MKN45 and NCI-N87 GC orthotopic implantations followed by treatment with AT1R blockers in MKN45 and NCI-N87 tumor-bearing athymic mice (created by BioRender). ( B) Mice transplanted with MKN45 and NCI-N87 cells show visible liver metastatic nodules. ( C) Orthotopic GC tumors show high expression of ATII and AT1R ( n = 11 per group). Original magnification, x20. ( D) Immunohistochemistry shows a significant change in expression of KLF4 in MKN45 and NCI-N87 orthotopic tumors, which are highly metastatic to the liver. Strong KLF4 expression is observed in normal mouse stomach tissues ( n = 11). Original magnification, x20. (E) In vivo administration of AT1R blockers, losartan and candesartan, for 28 consecutive days significantly reduces tumor growth in MKN45 and NCI-N87 orthotopic xenograft models, as measured by the wet tumor weight. Each dot represents a mouse. Data represented as mean±SEM. Significance was determined by 1-way ANOVA with Dunnett’s multiple comparisons test. (F) Treatment with losartan and candesartan also results in a significant reduction in distant metastasis. (G) qRT-PCR analysis shows significant upregulation of KLF4, CLDN1, CLDN3, CLDN4, and TJP1 transcripts in GC tissues from losartan-treated mice compared to those from untreated mice. For the quantification of mRNA levels, genes were normalized to the housekeeping gene GAPDH. Data represented as mean±SD and analyzed using 1-way ANOVA with Turkey’s multiple comparisons test (n = 3). ( H) Western blot analysis further confirms the significant increase in the expression of KLF4, Claudin 1 and Claudin 4 in mouse GC tissues upon losartan treatment. *P < 0.05 ; **P < 0.01 ; ***P < 0.001 ; ****P < 0.0001. GC, gastric cancer; IHC, immunohistochemistry; TJ, tight junction.

Article Snippet: The antibodies ATII (Cat# NB100-62346 Novus Biologicals, CO), AT1R (Cat# ab124505 Abcam, MA), KLF4 (Cat# ab216968 Abcam, MA and Cat# 4038S Cell Signaling Technology, Danvers, MA), claudin 1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), claudin 3 (Cat# NBP2-46299 Novus Biologicals, CO), claudin 4 (Cat# ab53156, Abcam, MA), Zonula occludens 1 (Cat# NBP2-80141 Novus Biologicals, CO), GAPDH (Cat#2118 Cell Signaling Technology, MA), HDAC1 (Cat# NB100-56340 Novus Biologicals, CO) were used for western blotting ( ).

Techniques: Expressing, Immunohistochemistry, In Vivo, Quantitative RT-PCR, Western Blot

Journal: Cancers

Article Title: A Tunable Nanoplatform of Nanogold Functionalised with Angiogenin Peptides for Anti-Angiogenic Therapy of Brain Tumours

doi: 10.3390/cancers11091322

Figure Lengend Snippet: ( a – c ) Ultraviolet (UV)-visible spectra of gold nanopartilces (AuNPs) in the 1 mM 3-(N-morpholino)propanesulfonic acid)-Tris(2-carboxyethyl)phosphine hydrochloride (MOPS-TCEP) buffer (1:1 mol ratio) before and after the addition of: ( a ) 30 μM Ang 60–68 , ( b ) 30 μM Ang 60–68 Cys; ( c ) 100 nM angiogenin (ANG). ( d ) UV-visible spectra of the pellets collected after two rinsing steps by centrifugation (15 min at 6010 relative centrifugal force, RCF) and re-suspension in 1 mM MOPS-TCEP buffer.

Article Snippet: After that, membranes were incubated with primary anti-angiogenin antibody (code: sc-9044, 1:500 dilution, from Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C.

Techniques: Centrifugation, Suspension

Journal: Cancers

Article Title: A Tunable Nanoplatform of Nanogold Functionalised with Angiogenin Peptides for Anti-Angiogenic Therapy of Brain Tumours

doi: 10.3390/cancers11091322

Figure Lengend Snippet: Confocal micrographs of A172 cells. Actin Green ® 488 (in green, ex/em= 488/500–530 nm) and Hoechst33342 (in blue, ex/em=405/425–475 nm) were used as F-actin and nuclear markers, respectively. Antibody against angiogenin shows angiogenin localisation in red (ex/em=543/560–700 nm) and micrographs are merged with optical bright field images (in grey). Before treatments, cell were rinsed with fresh culture medium and incubated for 2 h with basal culture medium (control: CTRL) and in culture medium supplemented with: Ang 60–68 (30 μM), Ang 60–68 Cys (30 μM); ANG (100 nM), AuNP (9.4 nM = 1.4 × 10 8 NP/mL), Ang 60–68 _NP (1.4 nM = 4.0 × 10 6 NP/mL, [Ang 60–68 ] = 2.8 × 10 −12 M), Ang 60–68 Cys_NP (1.4 nM = 4.0 × 10 6 NP/mL, [Ang 60–68 Cys] = 2.6 × 10 −12 M), ANG_NP (1.2 nM = 3.4 × 10 6 NP/mL, [ANG]= 0.2 × 10 −12 M). Scale bar = 10 μm.

Article Snippet: After that, membranes were incubated with primary anti-angiogenin antibody (code: sc-9044, 1:500 dilution, from Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C.

Techniques: Incubation, Control

Journal: Cancers

Article Title: A Tunable Nanoplatform of Nanogold Functionalised with Angiogenin Peptides for Anti-Angiogenic Therapy of Brain Tumours

doi: 10.3390/cancers11091322

Figure Lengend Snippet: Confocal micrographs of d-SH-SY5Y cells Actin Green ® 488 (in green, ex/em = 488/500–530 nm) and Hoechst33342 (in blue, ex/em = 405/425–475 nm) were used as F-actin and nuclear markers, respectively. Antibody against angiogenin shows angiogenin localisation in red (ex/em=543/560–700 nm) and micrographs are merged with optical bright field images (in grey). Before treatments, cell were rinsed with fresh culture medium and incubated for 2 h with basal culture medium (control: CTRL) and in culture medium supplemented with: Ang 60–68 (30 μM), Ang 60–68 Cys (30 μM); ANG (100 nM), AuNP (9.4 nM = 1.4 × 10 8 NP/mL), Ang 60–68 _NP (1.4 nM = 4.0 × 10 6 NP/mL, [Ang 60–68 ] = 2.8 × 10 −12 M), Ang 60–68 Cys_NP (1.4 nM = 4.0 × 10 6 NP/mL, [Ang 60–68 Cys] = 2.6 × 10 −12 M), ANG_NP (1.2 nM = 3.4 × 10 6 NP/mL, [ANG]= 0.2 × 10 −12 M). Scale bar = 10 μm.

Article Snippet: After that, membranes were incubated with primary anti-angiogenin antibody (code: sc-9044, 1:500 dilution, from Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C.

Techniques: Incubation, Control

Journal: Hypertension in pregnancy

Article Title: CircYTHDF1/miR-19b-3p/YTHDF1 axis contributes to pregnancy-induced hypertension development by enhancing vascular endothelial cell injury.

doi: 10.1080/10641955.2024.2414976

Figure Lengend Snippet: Figure 1. The inflammatory cytokines and biochemical parameters are elevated in the peripheral blood of PIH patients. The peripheral blood of PIH patients and healthy subjects were collected, and ELISA was employed to detect the level of IL-1β (A), TNF-α (B), IL-6 (C), TGF-β1 (D), ET-1 (E) and Ang-II (F). ***p < 0.001 vs Normal.

Article Snippet: The level of TGF-β1, TNF-α, IL-6, IL-1β, ET-1, and Ang-II in the peripheral blood of PIH patients and healthy subjects were measured using Human TGF-β1 ELISA Kit (E-EL0162c, Elabscience, USA), Human TNF-α ELISA Kit (E-EL-H0109c, Elabscience), Human IL-6 ELISA Kit (E-EL-H6156, Elabscience), Human IL-1β ELISA Kit (E-EL-H0149c, Elabscience), Human ET-1 ELISA Kit (E-EL-H0064c, Elabscience), and Human Ang-II ELISA Kit (E-EL-H0326c, Elabscience), respectively, according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Hypertension in pregnancy

Article Title: CircYTHDF1/miR-19b-3p/YTHDF1 axis contributes to pregnancy-induced hypertension development by enhancing vascular endothelial cell injury.

doi: 10.1080/10641955.2024.2414976

Figure Lengend Snippet: Figure 2. CD4+ T cells isolated from the peripheral blood of PIH patients promotes HUVEC apoptosis. CD4+ T cells were isolated from the peripheral blood of PIH patients and healthy subjects, and which were co-cultured with HUVEC. ELISA was employed to detect the level of Ang-II (A) and ET-1 (B) in the supernatant of HUVEC; flow cytometry was employed to detect HUVEC apoptosis (C); western blot was performed to detect the protein level of GPX4, FSP, and CoQ10B in HUVEC (D). Western blot (E) and qRT-PCR (F–H) were used to examine the protein and mRNA levels of KEAP1, NRF2, and HO-1 in HUVEC. **p < 0.01, ***p < 0.001 vs Normal.

Article Snippet: The level of TGF-β1, TNF-α, IL-6, IL-1β, ET-1, and Ang-II in the peripheral blood of PIH patients and healthy subjects were measured using Human TGF-β1 ELISA Kit (E-EL0162c, Elabscience, USA), Human TNF-α ELISA Kit (E-EL-H0109c, Elabscience), Human IL-6 ELISA Kit (E-EL-H6156, Elabscience), Human IL-1β ELISA Kit (E-EL-H0149c, Elabscience), Human ET-1 ELISA Kit (E-EL-H0064c, Elabscience), and Human Ang-II ELISA Kit (E-EL-H0326c, Elabscience), respectively, according to the manufacturer’s instructions.

Techniques: Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR