Fig. 6: Homophilic ATP1A1 interactions promote activin A secretion.
a ELISA analysis of activin A in fibroblasts generated from two distinct individuals (PSC and PSC-2) after direct contact with purified tumor membrane proteins (from BxPC-3, SU.86.86, MIA PaCa-2, or PANC-1 cells). Values were presented as mean ± SD (n = 3). b Western blotting analysis of no cross-linking control and cross-linked membrane protein complexes with streptavidin-HRP under reducing condition. Since 2-mercaptoethanol broke disulfide bond on the cross-linker, a band (*) of biotin-labeled tumor membrane proteins from BxPC-3 and SU.86.86 cells were identified. c Five plasma membrane proteins were identified by mass spectrometry analysis. d ELISA analysis of activin A in mono-cultured or direct co-cultured PSCs with BxPC-3 cells stably expressing lentiviral-based LacZshRNA, ATP1A1shRNA Clone#1, ATP1A2shRNA, ATP1A3shRNA, EMC1shRNA Clone#1, ITGB1shRNA. Values were presented as mean ± SD (n = 3). e ELISA analysis of activin A in fibroblasts co-cultured with 2% paraformaldehyde fixed BxPC-3 cells and treated with IgG (4 μg/ml), anti-ATP1A1-neutralizing antibody (2 and 4 μg/ml, 14418-1-AP, Proteintech), or anti-EMC1-neutralizing antibody (2 and 4 μg/ml, GTX119884, Genetex). Values were presented as mean ± SD (n = 3). f Left panel, western blotting analysis of biotinylated fibroblast membrane proteins with streptavidin-HRP under reducing condition. A band (*) of biotin-labeled fibroblast membrane proteins which bound to ATP1A1 was identified. Right panel, re-probing the western blot membrane from left panel with anti-Flag antibody to confirm that the identified band (*) was not ATP1A1-Flag. g ATP1A1 was identified by mass spectrometry analysis. h ELISA analysis of activin A in mono-cultured and direct co-cultured BxPC-3 cells with PSCs stably expressing lentiviral-based LacZshRNA and ATP1A1shRNA Clone#1. Values were presented as mean ± SD (n = 3). i Immunofluorescence staining of ATP1A1 in fibroblasts (PSC-GFP) and cancer cells (BxPC-3-RFP and SU.86.86-RFP) measured by confocal microscope. Scale bar, 2 μm. j Live-cell confocal imaging of BxPC-3 cells stably expressing ATP1A1-GFP and PSCs stably expressing ATP1A1-RFP. Co-localization of ATP1A1 was compared between initiation and establishment of tumor-fibroblast contact within 15 mins. Scale bar, 5 μm. k, l Correlation of ATP1A1 and INHBA expression levels in PDAC patient specimen. k Representative images of ATP1A1 expression (brown color, IHC) and INHBA expression (activin A, pink, in situ hybridization, RNAscope). Scale bar, 25 μm. l Pearson’s correlation coefficient analysis of ATP1A1 and INHBA according to H score and tumor-adjacent INHBA+ cells (%) (n = 100, r = 0.52, p < 0.0001). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).
