Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular matrix protein signaling promotes multi-step cancer vasculogenic mimicry formation

doi: 10.1186/s12964-025-02428-0

Figure Lengend Snippet: Integrin ß1, but not integrin ß3, is required for the formation of VM HEY-A8 GFP ( A ) or MDA-MB-231 ( B ) cells were incubated with a blocking antibody against integrin β1 or integrin β3 before seeding on Matrigel for 96 h. Photographs were taken at 24 and 96 h. Blocking of integrin β1 is enough to stop the formation of VM. ( C ) Airy Scan microscopy demonstrating the presence of integrin ß1 (red) and the cell nucleus stained by DAPI (blue) in both cell lines during VM formation at 2 and 72 h. Scale bar = 100 μm. Panel A &B, representative images from a minimum of three experiments in each cell line. Panel C representative images from two experiments

Article Snippet: 3-D Culture Matrix Rat Collagen I (3447-020-01) and 3-D Culture Matrix Laminin I (3446-005-01) were purchased from R&D Systems (Minneapolis, MN). siRNA against Integrin β1 (sc-35674), primary antibodies Integrin β1 (P5D2, sc-13590), Cortactin (H-5, sc-55579) and Integrin β3 (2C9.G2, sc-46655) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Incubation, Blocking Assay, Microscopy, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular matrix protein signaling promotes multi-step cancer vasculogenic mimicry formation

doi: 10.1186/s12964-025-02428-0

Figure Lengend Snippet: Integrin ß1 is essential for the formation of VM HEY-A8 ( A ) or MDA-MB-231 ( B ) cells were transfected with siScrambled or siRNA against integrin β1 (siIntegrin ß1) before seeding on Matrigel for 96 h. Photographs were taken at 24 and 96 h. Cells that were transfected by siIntegrin β1 do not form VM. ( C )-( E ) Western blot demonstrates that levels of integrin ß1 protein were reduced. * = p < 0.05. Scale bar on panel A = 100 μm. Representative images from a minimum of three experiments in each cell line

Article Snippet: 3-D Culture Matrix Rat Collagen I (3447-020-01) and 3-D Culture Matrix Laminin I (3446-005-01) were purchased from R&D Systems (Minneapolis, MN). siRNA against Integrin β1 (sc-35674), primary antibodies Integrin β1 (P5D2, sc-13590), Cortactin (H-5, sc-55579) and Integrin β3 (2C9.G2, sc-46655) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Transfection, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Extracellular matrix protein signaling promotes multi-step cancer vasculogenic mimicry formation

doi: 10.1186/s12964-025-02428-0

Figure Lengend Snippet: Integrin β1 is highly expressed and localized on the plasmatic membrane during VM formation A and B Confocal microscopy showing that integrin β1 is localized in the plasmatic membrane of HEY-A8 cells at 2 h of VM formation. ( C ) Quantification of integrin β1 intensity comparing cells grown on denatured Matrigel (control) and cells grown onto Matrigel after 2 h (VM). D HEY-A8 cells were seeded onto Matrigel and fixed at different timepoints. Confocal microscopy shows that integrin β1 is highly localized at 2 h of VM formation but not at 8 and 24 h. E Quantification of integrin β1 intensity comparing different timepoints of VM formation. F 3D reconstruction of z-stacks showing that integrin β1 is localized at the top of the VM tube at 96 h. G Confocal microscopy showing that integrin β1 is more present at the top of the VM tubular structure compared to the bottom and the middle of the tube. For all images integrin β1 (red), F-actin (green) and the cell nucleus stained by DAPI (blue). Representative images from a minimum of three experiments in each cell line. * = p < 0.05

Article Snippet: 3-D Culture Matrix Rat Collagen I (3447-020-01) and 3-D Culture Matrix Laminin I (3446-005-01) were purchased from R&D Systems (Minneapolis, MN). siRNA against Integrin β1 (sc-35674), primary antibodies Integrin β1 (P5D2, sc-13590), Cortactin (H-5, sc-55579) and Integrin β3 (2C9.G2, sc-46655) were purchased from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Membrane, Confocal Microscopy, Control, Staining

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: Integrin β1 downregulation decreased ACE2 expression in human renal epithelial cells. A, B) Human renal proximal tubule cell HK-2 was treated with integrin β1 shRNA. The expression of integrin β1 was detected with immunoblotting using integrin β1 antibody (Cell Signaling, 9699) that showed a significant reduction in the presence of integrin β1 shRNA compared to control shRNA treated cells (# p < 0.05, n = 3). C) Total mRNA quantified by qRT-PCR showed a significant reduction in integrin β1 shRNA treated cells compared to control shRNA treated cells (# p < 0.05, n = 3). D, E) The surface expression of integrin β1 was estimated using Flow cytometry analysis revealed a significant decrease in the median fluorescent intensity. Integrin β1 was stained with primary antibody (Abcam, ab 179471) and BV421-conjugated anti-rabbit secondary antibody (BD Bioscience, 56501) (# p < 0.001, n = 3). F, G) ACE2 expression decreased significantly in integrin β1 shRNA-treated HK-2 cells compared to control shRNA-treated cells in western blotting (ACE2 antibody, Thermo Fisher, MA5–32307) (# p < 0.05, n = 3). H) ACE2 mRNA as quantified by qRT-PCR also showed a significant reduction in integrin β1 shRNA treated cells compared to control shRNA treated cells (# p < 0.05, n = 3).

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Expressing, shRNA, Western Blot, Quantitative RT-PCR, Flow Cytometry, Staining

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: Pharmacological inhibition of integrin β1 using BTT 3033 decreased ACE2 expression in HK-2 cells. A) HK-2 cells were treated with DMSO and integrin α2β1 inhibitor BTT 3033. Treated cells were immune-stained for active integrin β1 (Millipore Sigma, MAB2247, clone 12G10). Total integrin β1 expression was monitored by integrin β1 antibody (Abcam, ab 179471). Scale bar 20 μm. Nucleus was stained with DAPI. B-C) Quantification of active integrin β1 shows a significant decrease in BTT 3033 treatment compared to DMSO in HK-2 Cells (n = 20, * p < 0.05) and no change in total integrin β1 expression with BTT 3033 treatment. D) mRNA level of ACE2 in BTT 3033 treated HK-2 cells was significantly lower compared to DMSO treated cells (# p < 0.05, n = 3). No significant change in the integrin β1 mRNA level was observed. E, F) Immunoblotting for integrin β1 (9699; Cell Signaling) did not show changes in protein level. G) Immunoblotting confirmed the decreased protein level expression of ACE2 (MA5–32307; Thermo Fisher).

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Inhibition, Expressing, Staining, Western Blot

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: Integrin β1 and ACE2 expression are higher in renal tumors compared to normal control samples. A) Gene expression of integrin β1 in renal tumors was compared to normal tissues using TCGA data and USCF Xena software. Integrin β1 levels were significantly higher in renal tumors (normal n = 160, tumor n = 170, p < 0.05). B, C) Immunoblotting of integrin β1 (Cell Signaling, 9699;) in HK-2 and Caki-1 cell line (n = 3) also showed higher integrin β1 expression in renal cancer (Caki-1) cells compared to epithelial cells (HK-2). D) Fold change in integrin β1 mRNA levels by qRT-PCR also shows higher integrin β1 levels in Caki-1 cells compared to renal epithelial cells (HK-2) (n = 3, # p < 0.05). E) TCGA data analysis showed significantly higher gene expression of ACE2 in renal tumors compared to normal tissues (normal n = 27, tumor n = 358, p < 0.05). F-G) Immunoblotting of ACE2 (Thermo Fisher, MA5–32307) normalized with GAPDH (Cell Signaling, 5174) also showed significantly higher expression of ACE2 in Caki-1 cells compared to renal epithelial cells (HK-2) (# p < 0.05, n = 3). H) ACE2 mRNA levels by qRT-PCR also showed higher ACE2 levels in Caki-1 cells compared to renal epithelial cells (HK-2) (# p < 0.05, n = 3).

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Expressing, Software, Western Blot, Quantitative RT-PCR

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: A decrease in the integrin β1 activation lowered ACE2 expression in Caki-1 cells. A) DMSO and BTT 3033 treated Caki-1 cells were stained for active integrin β1 (Millipore Sigma, MAB2247, clone 12G10). Total integrin β1 expression was monitored by integrin β1 antibody (Abcam, ab 179471). Scale bar 20 μm. B-C) Active integrin β1 shows a significant decrease in BTT 3033 treated Caki-1 cells compared to DMSO treated cells (n = 20, # p < 0.05) and no change in total integrin β1 expression with BTT 3033 treatment. D) Fold changes in mRNA level of integrin β1 and ACE2 quantified by qRT-PCR showed a significant reduction in ACE2 mRNA levels in BTT 3033 treated cells compared to DMSO treated cells. Integrin β1 mRNA levels did not alter significantly by BTT 3033 treatment. E-G) immunoblotting using integrin β1(Cell Signaling, 9699) and ACE2 (Thermo Fisher, MA5–32307) also confirmed the lower ACE2 levels in BTT 3033 treated cells compared to DMSO treated cells (# p < 0.05, n = 3).

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Activation Assay, Expressing, Staining, Quantitative RT-PCR, Western Blot

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: SARS-CoV-2 spike protein internalization decreased in the presence of BTT 3033 in HK-2 and Caki-1 cells. A) Immunoblotting using an anti-His antibody (Santa Cruz Biotechnology, SC53073) for control and SARS-CoV-2 spike protein after 2 h of treatment with DMSO and BTT 3033 in HK-2 cells. GAPDH was used as a loading control (5174; Cell Signaling). B) No change in control protein internalization in DMSO and BTT 3033 treatment groups. Protein quantification shows a significantly lower SARS-CoV-2 spike protein internalization in BTT 3033 treated HK-2 cells (# p < 0.05, n = 3) compared to DMSO-treated cells. C) SARS-CoV-2 spike protein internalization was decreased in Caki-1 in the presence of BTT 3033. Caki-1 cell lysates were immunoblotted using an anti-His antibody for control and SARS-CoV-2 spike protein after 2 h of treatment with DMSO and BTT 3033 in HK-2 and Caki-1 cells. D) Normalized protein quantification shows a significantly lower SARS-CoV-2 spike protein internalization in BTT 3033 treated Caki-1 cells (# p < 0.05, n = 3) and no change in control protein internalization. E, F) HK-2 and Caki-1cells lysates were analyzed for the internalized spike protein using ELISA. There was a significant reduction in internalized protein in presence of BT 3033 (# p < 0.001, n = 3). G) Control and integrin β1 shRNA-treated HK-2 cells were compared to estimate the amount of spike protein internalized. There was a significant reduction in internalized spike protein in the integrin β1 shRNA-treated cells compared to control shRNA-treated cells (# p < 0.001, n = 3).

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, shRNA

Journal: European journal of cell biology

Article Title: Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells

doi: 10.1016/j.ejcb.2023.151316

Figure Lengend Snippet: Deleting integrin β1 from the kidney proximal tubules epithelial cells decreased ACE2 expression. A, B) Renal proximal tubule-specific depletion of integrin β1 was carried out using a ɣgt cre. The decreased expression of integrin β1 was confirmed by immunoblotting of cortical lysate integrin β1 (Santa Cruz Biotechnology, SC-374430). Integrin β1 KO mice showed a significant decrease in expression compared to integrin β1 WT mice (# p < 0.05, n = 3). C) The epithelial cell-specific deletion was further confirmed by immunofluorescence. LTL (green) indicates the proximal tubules. Integrin β1(Abcam, ab179471) (red) staining was significantly reduced in LTL-positive cells. A, B) Immunoblotting shows a significantly lower ACE2 (Invitrogen, MA532307) in the kidney cortex of integrin β1 KO mice compared to WT mice (# p < 0.05, n = 3). D) mRNA analysis by qRT-PCR confirmed the decreased ACE2 expression. E) ACE2 expression in the proximal tubules was investigated using immune fluorescence. Proximal tubule marker LTL (green) was also used to counter-stain. Representative results from 3 independent mice kidneys show lower ACE2 (red) in the proximal tubules of the integrin β1 KO mice compared to WT mice.

Article Snippet: Primary antibody for active integrin β1 (Dilution: 1:250; Cat: MAB2247, clone 12G10; Millipore Sigma) and total integrin β1 (Dilution: 1:250; Abcam, ab179471) was incubated at 4°C.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Quantitative RT-PCR, Fluorescence, Marker