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 and B) The qRT-PCR data show that blocking AT1R with losartan over time, from 2 hours to 24 hours in MKN45 and NCI-N87 GC cells, significantly increases the expression of TJ genes, CLDN1, CLDN3, CLDN4, and TJP1 . For quantification, the mRNA level of TJ genes was normalized to the housekeeping gene GAPDH. Data represented as mean±SD and analyzed using 2-way ANOVA with Dunnett’s multiple comparisons test ( n = 3). ( C and D) Western blot data also confirm the increase in the expression of TJ proteins in GC cells upon AT1R blockade. ( E and F) Immunofluorescence staining further demonstrates the enhanced membrane localizations of Claudin 1, Claudin 4, and Zonula occludens 1 in losartan-treated GC cells compared to untreated controls. Original magnification, ×20. The green fluorescence intensity in maximum projection images of Z-stacks was quantified. The green channel was thresholded to create binary masks encompassing all cells, while excluding background, followed by measurement of the mean intensity in arbitrary fluorescence units, as shown in the bar diagram. Data represented as mean±SEM of three independent experiments and analyzed using 2-tailed t test. * P < 0.05; ** P <0.01;*** P < 0.001; **** P < 0.0001. ns : no significance. 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: Quantitative RT-PCR, Blocking Assay, Expressing, Western Blot, Immunofluorescence, Staining, Membrane, Fluorescence

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) Western Blot analysis shows reduced expression of multiple TJ proteins, including Claudin 1, 3, 4, and Zonula occludens 1, in KLF4KOMKN45 and KLF4KONCI-N87 GC cells compared to scramble cells. ( B) qRT-PCR analysis indicates significant downregulation of TJ genes, CLDN1, CLDN3, CLDN4, and TJP1 at the transcript level in both KLF4KOMKN45 and KLF4KONCI-N87 cells. The quantification of mRNA levels of TJ genes was normalized to the housekeeping gene GAPDH. Data represented as mean±SD and analyzed using 1-way ANOVA with Dunnett’s multiple comparisons test and 2-tailed t test (n = 3) ( C) Representative confocal images of immunofluorescence staining for TJ proteins in scrambled control and KLF4KOMKN45 cells. DAPI (Blue) is used as a nuclear stain. Original magnification, x20. Loss of membrane-localized TJ proteins is evident in KLF4KO cells, indicating disruption of junctional integrity. Insets show higher magnification of the selected regions. The green fluorescence intensity in maximum projection images of Z-stacks was quantified. The green channel was thresholded to create binary masks encompassing all cells, while excluding the background, followed by measurement of the mean intensity in arbitrary fluorescence units, as shown in the bar diagram. Data represented as mean±SEM of three independent experiments and analyzed using 2-tailed t test. ( D) ChIP followed by PCR demonstrates the direct binding of KLF4 to the promoter regions of CLDN1 , CLDN3 , CLDN4 , and TJP1 , supporting the transcriptional regulation of TJ genes by KLF4. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. GC, gastric cancer; TJ, tight junction; ChIP, chromatin immunoprecipitation; Scr, scrambled control.

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: Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Control, Membrane, Disruption, Fluorescence, Binding Assay, Chromatin Immunoprecipitation

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 and B) Wound closure and transwell assay demonstrate that losartan treatment does not significantly alter the migratory and invasive potential of KLF4KO MKN45 and KLF4KONCI-N87 cells compared to untreated KLF4KOMKN45 and KLF4KONCI-N87 cells (magnification migration, x4; magnification invasion, x20) ( C) The percentage of wound closure is calculated and shown as a bar diagram. Data represented as mean±SEM. Significance was determined by 2-tailed t test ( n = 12). The cell invasive index was analyzed by counting the number of cells per microscopic field and is shown as a bar diagram. Data represented as mean±SEM. Significance was determined by 2-tailed t test ( n = 9). ( D) Western blot analysis demonstrates that there is no significant change in the expression levels of TJ proteins, Claudin 1, 3, 4, and Zonula occludens 1, in losartan-treated KLF4KOMKN45 and KLF4KONCI-N87 cells compared to untreated knockout cells. ns : no significance. GC, gastric cancer; TJs, tight junctions.

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: Transwell Assay, Migration, Western Blot, Expressing, Knock-Out

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: Representative western blot data show the expression levels of Claudin 1, 3, 4, and Zonula occludens 1 in normal mouse stomach tissue. GAPDH was used as a loading control. 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: Western Blot, Expressing, Control