Journal: Cell Death Discovery

Article Title: Targeting the hERG1/β1 integrin complex in lipid rafts potentiates statins anti-cancer activity in pancreatic cancer

doi: 10.1038/s41420-025-02321-2

Figure Lengend Snippet: A Representative blot (top) and densitometric analysis (bottom) of Rac-1 activation assay in PANC-1 cells untreated (CTR) or treated with MβCD (5 mM), scDb-hERG1-β1 (20 µg/ml) and their combination, seeded on FN for 90 min. GDP was used as negative control and GTPγS was used as positive control. Data are presented as mean values ± s.e.m. ( n = 3). a.u. = arbitrary units. Membranes were probed with Rac-1 antibody. Inputs of total Rac-1 and tubulin are reported in the figure. B IF images of PANC-1 cells untreated (CTR) or treated with MβCD (5 mM), scDb-hERG1-β1 (20 µg/ml) and their combination, seeded on FN for 90 min, stained with anti-ARP2/3 antibody and Cortical F actin (left panels). Scale bar: 100 µm. At least 20 cells (in 3 different fields) per condition from three independent experiments ( n = 3) were analyzed. Quantification graphs of ARP2/3 fluorescent intensity and cortical F-actin density were reported in the right panels. (ARP2/3 Fluorescence intensity) MβCD vs scDb-hERG1-β1: p = 0.02. scDb-hERG1-β1 vs MβCD+scDb-hERG1-β1: p = 0.01. (Cortical F-actin density) MβCD vs scDb-hERG1-β1: p = 0.04. scDb-hERG1-β1 vs MβCD+scDb-hERG1-β1: p = 0.02. C Lateral motility experiments onto FN were performed on PANC-1 cells treated with MβCD (5 mM), scDb-hERG1-β1 (20 μg/ml) and their combination onto FN for 90 min. Representative images are reported in the left panel. The motility is reported as graph of percentage of cell motility in the right panel. Scale bar: 100 µm. Data are presented as mean values ± s.e.m. ( n = 3). ( D ) Representative blot (top) and densitometric analysis (bottom) of Cyclin D, Cyclin E and p21 in PANC-1 cells untreated (CTR) or treated with MβCD (5 mM), scDb-hERG1-β1 (20 µg/ml) and their combination, seeded on FN for 90 min and a negative control, labeled BSA. Data are presented as mean values ± s.e.m. ( n = 3). a.u. = arbitrary units. Membranes were probed with anti-Cyclin D, anti-Cyclin E and anti-p21 antibodies. E Flow cytometry (FC) plots of cell cycle of PANC-1 cells treated with MβCD (5 mM), scDb-hERG1-β1 (20 μg/ml) and their combination for 24 h. MβCD vs CRT: p = 0.005 (G1); p = 0.042 (S); p = 0.041 (G2/M). scDb-hERG1-β1 vs CRT: p = 0.005 (G1); p = 0.038 (S); p = 0.039 (G2/M). MβCD+scDb-hERG1-β1 vs CTR: p = 0.0003 (G1); p = 0.001 (S); p = 0.0002 (G2/M). Data are presented as mean values ± s.e.m. ( n = 3). F Integrin controlled macromolecular hubs centered on hERG1 and lipid rafts and their possible involvement in pancreatic ductal adenocarcinoma. Created with BioRender.com. * P < 0.05; ** P < 0.01, and *** P < 0.001 (one-way ANOVA). GDP guanosine diphosphate, GTPγS G-protein-activating analog of guanosine triphosphate, CTR control, MβCD Methyl-β-cyclodextrin, MOC Mander’s Overlapping Coefficient, IP immunoprecipitation, BSA bovine serum albumin.

Article Snippet: The following primary antibodies were used: anti-phospho AKT (Thr 308) (pAKT) r-pAb (Cell Signaling Technology, Massachusetts, Danvers, USA, cat. BK9275S) or anti-total-AKT (t-AKT) r-pAb (Cell Signaling Technology Massachusetts, Danvers, USA, cat. BK9272S) at final dilution 1:500 for WB; anti-phospho ERK1/2 (p-ERK1/2) (Thr202/tyr 204) r-mAb (Cell Signaling Technology Massachusetts, Danvers, USA, cat. 4370) at final dilution 1:1000 for WB; anti-total ERK1/2 (t-ERK1/2) pAb (Santa Cruz Biotechnology, Santa Cruz, CA, cat. (1-C16)sc93) at final dilution 1:200 for WB; r-pAb anti β1-integrin, RM-12 (Immunological Science, Rome, Italy) at final dilution 1:1000 for WB; r-pAb anti-hERG1, C54 (MCK Therapeutics Srl, Pistoia, Italy) at a final dilution 1:1000 for WB; m-mAb hERG1 (MCK Therapeutics Srl, Pistoia, Italy) 5 µg antibody/mg protein for Co-IP; the Alexa 488 conjugated mAb hERG1 was used at 1 μg/ml for FACS experiments (MCK Therapeutics Srl, Pistoia, Italy); m-mAb β1 TS2/16 (Ultra-LEAF™ Purified anti-human CD29 Antibody, Bio Legend, cat. 303035) 5 µg antibody/mg protein for Co-IP, 1:500 for IF; scDb-hERG1/β1 (Single chain diabody, MCK Therapeutics Srl, Pistoia, Italy), 20 μg/ml for cell treatment and for immunohistochemistry (IHC); scDb-hERG1/β1-alexa-488 20 μg/ml for IF; m-mAb Anti-α-Tubulin (Sigma-Aldrich cat. T9026) at 1:500 dilution for WB; anti-PIP2 (phosphatidylinositol 4,5-bisphophate) 1:200 for IF (MA3-500 Invitrogen, Waltham, MA, USA); anti-PIP3 (phosphatidylinositol 3,4,5-bisphophate) 1:100 for IF (A21328 Invitrogen, Waltham, MA, USA); anti-caveolin-1 pAb (Abcam, Cambridge, UK) at 1:1000 for WB. m-mAb Anti-caveolin-1 (610406, BD Biosciences, Franklin Lakes, NJ) at 1:1000 for WB and 1:50 for IF; m-mAb Anti-Flotillin-1 (sc-25506, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 for WB and 1:50 for IF, Anti-ARP2/3 complex (BS-12524R, Thermo Fisher Scientific, Waltham, MA) at 1:100 for IF.

Techniques: Activation Assay, Negative Control, Positive Control, Staining, Fluorescence, Labeling, Flow Cytometry, Control, Immunoprecipitation

Journal: Cell Death Discovery

Article Title: Targeting the hERG1/β1 integrin complex in lipid rafts potentiates statins anti-cancer activity in pancreatic cancer

doi: 10.1038/s41420-025-02321-2

Figure Lengend Snippet: A PANC-1 and HEK-hERG1 cells untreated or treated with SIM or MβCD were subject to cholesterol quantification by HPTLC. Quantitative analysis of separated free cholesterol was carried out using NIH Image1.62 as software. B IF performed on PANC-1 cells following 90 min adhesion onto FN with or without treatment with SIM, scDb-hERG1-β1 and their combination. Representative images (scale bar: 100 μm) of caveolin-1 staining, scDb-hERG1-β1 staining and colocalization between caveolin-1 and scDb-hERG1-β1 are on the top, while quantitative analyses and MOC are reported in the graphs on the bottom. a.u.= arbitrary units. At least 20 cells (in 3 different fields) per condition from three independent experiments ( n = 3) were analyzed. All data are presented as mean values ± s.e.m. C IF performed on PANC-1 cells untreated (CTR) or treated with SIM (4.7 μM), scDb-hERG1-β1 (20 µg/ml), and their combination, seeded on FN for 90 min. Representative images of PIP2 (top panels) and PIP3 (bottom panels) (scale bar: 50 μm) are on the top, while quantitative analyses (Mean fluorescence intensity) are reported in the graph on the bottom. At least 20 cells (in 3 different fields) per condition from three independent experiments ( n = 3) were analyzed. All data are presented as mean values ± s.e.m. D Representative blot (top) and densitometric analysis (bottom) of phospho-Akt and phospho-ERK levels in PANC-1 cells untreated (CTR) or treated with SIM (4.7 µM), scDb-hERG1-β1 (20 µg/ml) and their combination, seeded on FN for 90 min. Data are presented as mean values ± s.e.m. ( n = 3). a.u. = arbitrary units. Membranes were probed with anti-pAkt Thr308, anti-Akt Thr308, ERK1/2 (pERK1/2) (Thr202/tyr204) and anti-total ERK1/2 antibodies. E IF on PANC-1 cells stained with anti-ARP2/3 antibody and cortical F-actin (left panels) after treatment with SIM (4.7 μM), scDb-hERG1-β1 (20 µg/ml) and their combination onto FN for 90 min (scale bar: 100 μm). At least 20 cells (in 3 different fields) per condition from three independent experiments ( n = 3) were analyzed. Quantification graphs of ARP2/3 fluorescent intensity and cortical F-actin density were reported in the right panels. Data are presented as mean values ± s.e.m. F Representative blot (top) and densitometric analysis (bottom) of Cyclin D, Cyclin E and p21 in PANC-1 cells untreated (CTR) or treated with scDb-hERG1-β1 (20ug/ml), SIM (4.7 µM) and their combination, seeded on FN for 90 min. Data are presented as mean values ± s.e.m. ( n = 3). a.u. = arbitrary units. Membranes were probed with anti-Cyclin D, anti-Cyclin E and anti-p21 antibodies. * P < 0.05; ** P < 0.01, and *** P < 0.001 (one-way ANOVA). CTR control, MβCD Methyl-β-cyclodextrin, SIM simvastatin, CHOL Free cholesterol, TGs triglycerides, CEs cholesterol esters, MOC Mander’s Overlapping Coefficient.

Article Snippet: The following primary antibodies were used: anti-phospho AKT (Thr 308) (pAKT) r-pAb (Cell Signaling Technology, Massachusetts, Danvers, USA, cat. BK9275S) or anti-total-AKT (t-AKT) r-pAb (Cell Signaling Technology Massachusetts, Danvers, USA, cat. BK9272S) at final dilution 1:500 for WB; anti-phospho ERK1/2 (p-ERK1/2) (Thr202/tyr 204) r-mAb (Cell Signaling Technology Massachusetts, Danvers, USA, cat. 4370) at final dilution 1:1000 for WB; anti-total ERK1/2 (t-ERK1/2) pAb (Santa Cruz Biotechnology, Santa Cruz, CA, cat. (1-C16)sc93) at final dilution 1:200 for WB; r-pAb anti β1-integrin, RM-12 (Immunological Science, Rome, Italy) at final dilution 1:1000 for WB; r-pAb anti-hERG1, C54 (MCK Therapeutics Srl, Pistoia, Italy) at a final dilution 1:1000 for WB; m-mAb hERG1 (MCK Therapeutics Srl, Pistoia, Italy) 5 µg antibody/mg protein for Co-IP; the Alexa 488 conjugated mAb hERG1 was used at 1 μg/ml for FACS experiments (MCK Therapeutics Srl, Pistoia, Italy); m-mAb β1 TS2/16 (Ultra-LEAF™ Purified anti-human CD29 Antibody, Bio Legend, cat. 303035) 5 µg antibody/mg protein for Co-IP, 1:500 for IF; scDb-hERG1/β1 (Single chain diabody, MCK Therapeutics Srl, Pistoia, Italy), 20 μg/ml for cell treatment and for immunohistochemistry (IHC); scDb-hERG1/β1-alexa-488 20 μg/ml for IF; m-mAb Anti-α-Tubulin (Sigma-Aldrich cat. T9026) at 1:500 dilution for WB; anti-PIP2 (phosphatidylinositol 4,5-bisphophate) 1:200 for IF (MA3-500 Invitrogen, Waltham, MA, USA); anti-PIP3 (phosphatidylinositol 3,4,5-bisphophate) 1:100 for IF (A21328 Invitrogen, Waltham, MA, USA); anti-caveolin-1 pAb (Abcam, Cambridge, UK) at 1:1000 for WB. m-mAb Anti-caveolin-1 (610406, BD Biosciences, Franklin Lakes, NJ) at 1:1000 for WB and 1:50 for IF; m-mAb Anti-Flotillin-1 (sc-25506, Santa Cruz Biotechnology, Santa Cruz, CA) at 1:1000 for WB and 1:50 for IF, Anti-ARP2/3 complex (BS-12524R, Thermo Fisher Scientific, Waltham, MA) at 1:100 for IF.

Techniques: High Performance Thin Layer Chromatography, Software, Staining, Fluorescence, Control

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Ang1 induces ARP2/3 expression in colorectal cancer cells in vitro. ( a – c ) Western blotting of ARP2/3 expression in colorectal cancer (COLO320dm, SW620 and MC38) cells in the presence or absence of Ang1 (top panel). The intensity of ARP2/3 bands ( n = 3) were quantified and normalized against GAPDH using ImageJ and represented as a fold change (bottom panel).

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Expressing, In Vitro, Western Blot

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Ang1 presence is essential for ARP2/3 expression in the cancer cells in vivo. ( a ) RepreScheme 1 and ARP2/3 staining of tumour sections generated by intrasplenic injection of mouse colorectal (MC38) cancer cells into wild type (Ang1 WT) and Ang1 knockout (Ang1 KO) mice (left panel). D: Desmoplastic ring, L: Liver tissue, T: Tumour. ( b ) Represents the Correlation between Ang1 expression in the liver tissue and ARP2/3 expression in the tumour cells using Pearson correlation analysis.

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Expressing, In Vivo, Staining, Generated, Injection, Knock-Out

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: ARP2/3 mediates Ang1-driven cancer cell motility. ( a , b ) Western blotting of ARP2/3 expression in SW620 or COLO320dm cancer cells expressing shRNA-scrambled or shRNA-ARPC3. The right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). ( c , d ) Representative scratch assay in SW620 or COLO320dm cells expressing shRNA-scrambled or shRNA-ARPC3 upon exposure to Ang1. The right panels show the corresponding wound healing ratio shown in fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Western Blot, Expressing, shRNA, Wound Healing Assay

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: Tie2 mediates Ang1-dependent ARP2/3 expression in the cancer cells. ( a ) Representative images of coimmunostaining for showing ARP2/3 (green) and Tie2 (red) on FFPE tissue sections of CRCLM resected from chemonaïve patients. The bottom panel represents the cancer cells at the invading front that are in direct contact with hepatocytes. ( b ) Immunoblotting of ARP2/3 expression in MC38 colorectal cancer cells expressing shRNA-Scrambled or shRNA-Tie2. The right panel shows the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). ( c , d ) Left panels show the effect of Tie2 inhibitor (BAY-826) on ARP2/3 expression in MC38 and SW620 cancer cells upon Ang1 exposure, respectively. Right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Expressing, Western Blot, shRNA

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: PI3K/AKT pathway contributes to Ang1-dependent ARP2/3 expression. ( a ) Protein interaction networks generated using STRING database version 11 showing the interaction between Ang1 (Angpt1), TIE2 (Tek), PI3Ks (PI3K), Akt (AKT1) and ARP2/3 subunits. ( b , c ) Western blotting of ARP2/3 expression in exposed MC38 or SW620 cancer cells to recombinant Ang1 in the presence or absence of PI3K/AKT inhibitor (LY294002). Right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change ( n = 3). Data are presented as the mean ± SD. ns = Not significant.

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Expressing, Generated, Western Blot, Recombinant

Journal: Cancers

Article Title: Angiopoietin-1 Upregulates Cancer Cell Motility in Colorectal Cancer Liver Metastases through Actin-Related Protein 2/3

doi: 10.3390/cancers14102540

Figure Lengend Snippet: The molecular mechanism of Ang1 function in vessel co-opting CRCLM lesions. Schematic representation of key findings in the current study. The hepatocytes of vessel co-opting lesions express high levels of Ang1. The secreted Ang1 by hepatocytes interacts with the cancer cells through Tie2, which activates PI3K/AKT followed by ARP2/3 expression, respectively. Upregulation in ARP2/3 expression increases cancer cell motility and allows them to infiltrate the liver tissue to obtain blood supply by hijacking the pre-existing vessels (vessel co-option).

Article Snippet: We baked the section at 60 °C for 1 h and performed staining using the following antibodies: ARP2/3 1:300 (Bioss, Laval, QC, Canada, #bs-12524R) and Ang1 1:500 (Abcam, Waltham, MA, USA, # ab102015).

Techniques: Expressing