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: 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 sections were again rinsed with PBS and incubated with 2.5% normal horse serum (Vector Laboratories, CA) at room temperature for 20 minutes to block the nonspecific binding sites and were stained for 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), CLDN1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), CLDN3 (Cat# NBP2-46299 Novus Biologicals, CO), CLDN4 (Cat# ab53156 Abcam, MA), ZO-1 (Cat# NBP2-80141 Novus Biologicals, CO).

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 sections were again rinsed with PBS and incubated with 2.5% normal horse serum (Vector Laboratories, CA) at room temperature for 20 minutes to block the nonspecific binding sites and were stained for 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), CLDN1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), CLDN3 (Cat# NBP2-46299 Novus Biologicals, CO), CLDN4 (Cat# ab53156 Abcam, MA), ZO-1 (Cat# NBP2-80141 Novus Biologicals, CO).

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 sections were again rinsed with PBS and incubated with 2.5% normal horse serum (Vector Laboratories, CA) at room temperature for 20 minutes to block the nonspecific binding sites and were stained for 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), CLDN1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), CLDN3 (Cat# NBP2-46299 Novus Biologicals, CO), CLDN4 (Cat# ab53156 Abcam, MA), ZO-1 (Cat# NBP2-80141 Novus Biologicals, CO).

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 sections were again rinsed with PBS and incubated with 2.5% normal horse serum (Vector Laboratories, CA) at room temperature for 20 minutes to block the nonspecific binding sites and were stained for 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), CLDN1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), CLDN3 (Cat# NBP2-46299 Novus Biologicals, CO), CLDN4 (Cat# ab53156 Abcam, MA), ZO-1 (Cat# NBP2-80141 Novus Biologicals, CO).

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 sections were again rinsed with PBS and incubated with 2.5% normal horse serum (Vector Laboratories, CA) at room temperature for 20 minutes to block the nonspecific binding sites and were stained for 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), CLDN1 (Cat# H00009076-M01 Abnova, Taipei, Taiwan), CLDN3 (Cat# NBP2-46299 Novus Biologicals, CO), CLDN4 (Cat# ab53156 Abcam, MA), ZO-1 (Cat# NBP2-80141 Novus Biologicals, CO).

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