Fig. 3: Effects of MβCD and scDb-hERG1-β1 treatment on hERG1/β1 integrin complex in PDAC cells after cell adhesion on Fibronectin.

A Flow cytometry plots of hERG1 expression onto the plasma membrane in HEK-HERG1 and PANC-1 cells following 90 min adhesion onto FN with or without treatment with 5 mM MβCD for 20 min [48]. Values are expressed as mean fluorescence intensity of the area under the curve (MFI). Representative plots are on the left, while quantitative analyses are reported in the graphs on the right. a.u. = arbitrary units Data are mean values ± s.e.m. obtained from three independent experiments (n = 3). B Co-IP between hERG1 and β1 integrin on HEK-hERG1 and PANC-1 cells following 90 min adhesion onto FN with and without treatment with 5 mM MβCD and corresponding densitometric analysis. Total cell proteins were immunoprecipitated with anti-β1 integrin mAb (TS2/16). An IgG isotypic control was employed too. Left panel: representative WB of the co-IP; Right panel: densitometric analysis. The WBs relative to the inputs are in figure and in Supplementary Fig. 3. Data are representative of three independent experiments (n = 3). a.u.= arbitrary units. C IF performed on HEK-hERG1 and PANC-1 cells following 90 min adhesion onto FN with or without treatment with 5 mM MβCD. Representative images (scale bar: 100 μm) of scDb-hERG1-β1 staining is on the right. a.u.= arbitrary units. At least 20 cells (in 3 different fields) per condition from three independent experiments (n = 3) were analyzed. D HEK-hERG1 and PANC-1 cells following 90 min adhesion onto FN with or without treatment with 5 mM MβCD. Representative images (scale bar: 100 μm) of caveolin-1 staining and colocalization between scDb-hERG1-β1 and caveolin-1 are on the left, while quantitative analyses (fluorescent intensity and Mander’s Overlapping Coefficient, MOC) are reported in the graphs on the right. 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. E IF performed on PANC-1 cells following 90 min adhesion onto FN with or without treatment with scDb-hERG1-β1 (20 μg/ml). Representative images of colocalization between scDb-hERG1-β1 and caveolin-1 (scale bar: 100 μm) are on the left, while quantitative analyses (MOC) are reported in the graph on the right. 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. F Co-IP between hERG1 and PI3K p85 in PANC-1 cells untreated (CTR) or treated with MβCD (5 mM), scDb-hERG1-β1 (20ug/ml), and their combination, seeded on FN for 90 min. Total cell proteins were immunoprecipitated with anti-hERG1 mAb. Left panel: representative WB of the co-IP; Right panel: densitometric analysis. Total lysates indicated as “inputs” are reported in Supplementary Fig. 3. Data are representative of three independent experiments (n = 3). a.u. = arbitrary units. G IF performed on 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. 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. Lower magnification images are reported in Supplementary Fig. 3. H Representative blot (left) and densitometric analysis (right) of ERK and phospho-ERK levels in PANC-1 cells untreated (CTR) or treated with scDb-hERG1-β1 (20 µg/ml), MβCD (5 mM) 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-pAkt Thr308, anti-Akt Thr308, ERK1/2 (pERK1/2) (Thr202/tyr204) and anti-total ERK1/2 antibodies. CTR control, MβCD Methyl-β-cyclodextrin, MOC Mander’s Overlapping Coefficient, IP immunoprecipitation, BSA bovine serum albumin. All data are presented as mean values ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 (one-way ANOVA).