Fig. 4: Effects of MβCD and scDb-hERG1-β1 treatment on intracellular signaling triggered by cell adhesion on Fibronectin in PDAC cells. | Cell Death Discovery

Fig. 4: Effects of MβCD and scDb-hERG1-β1 treatment on intracellular signaling triggered by cell adhesion on Fibronectin in PDAC cells.

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

Fig. 4

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.

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