Fig. 7: Homophilic ATP1A1 interactions stabilize ATP1A1 in plasma membrane of fibroblasts trigger intracellular Ca²⁺ oscillations for turning on activin A secretion via NF-κB signaling.

a Western blotting analysis of plasma membrane proteins from intracellular hydrogelated PSCs and BxPC-3 cells. Before intracellular hydrogelation, cells were cultured with or without 2% paraformaldehyde fixed BxPC-3 cells or PSCs for 24 h. b Immunofluorescence staining of ATP1A1 in fibroblasts (PSC-GFP) and cancer cells (BxPC-3-RFP) upon mono-cultured or direct co-cultured conditions measured by confocal microscope. Scale bar, 10 μm. c Quantification results of ATP1A1 intensity analyzed by Image J. Values were presented as mean ± SD (n = 7). d Immunofluorescence staining of ATP1A1 in fibroblasts (PSC-GFP) and cancer cells (BxPC-3-RFP cells stably expressing lentiviral-based LacZ^shRNA or ATP1A1^{shRNA Clone#1}) upon mono-cultured or direct co-cultured conditions measured by confocal microscope. Scale bar, 10 μm. e Quantification results of ATP1A1 intensity analyzed by Image J. Values were presented as mean ± SD (n = 12). f The differential gene expression analysis with RNA-seq. Overlap of the commonly up-regulated genes (2992) in fiborblasts (PSC-GFP) after direct co-cultured (D) with BxPC-3 and SU.86.86 cells (but not MIA PaCa-2 and PANC-1 cells) compared to mono-cultured (−) or indirect co-cultured (ID) conditions were used for Ingenuity Pathway Analysis (IPA). Top 2 canonical pathways with the largest -log (p-value) calculated by Fisher’s Exact Test in IPA were shown. g Intracellular Ca²⁺ concentration of BxPC-3 cells and PSCs in mono-cultured or direct co-cultured conditions measured by fluorescence ratios (340/380 nm) using Fura-2/AM. Values were presented as mean ± SD (n = 20). h Intracellular Ca²⁺ concentration of PSCs cells upon mono-cultured or direct co-cultured with BxPC-3 cells stably expressing lentiviral-based LacZ^shRNA or ATP1A1^{shRNA clone#1} measured by fluorescence ratios (340/380 nm) using Fura-2/AM. Values were presented as mean ± SD (n = 20). i ELISA analysis of activin A in mono-cultured or direct co-cultured PSCs and BxPC-3 cells with Ca channel inhibitor (Nifedipine). Values were presented as mean ± SD (n = 3). j Western blotting analysis of proteins harvested from co-cultured fibroblast-GFP with BxPC-3 cells. FACS was performed to collect GFP⁺ and GFP^- cells after 48 hours of indirect or direct co-culture. k Western blotting analysis of proteins harvested from indirect- and direct co-cultured PSC cells with BxPC-3-RFP stably expressing lentiviral-based LacZ^shRNA and ATP1A1^{shRNA Clone#1}. FACS was performed to collect RFP^- cells after 48 hours of indirect (ID)- or direct (D)- co-cultured conditions. l ELISA analysis of activin A in mono-cultured or direct co-cultured PSCs and BxPC-3 cells with NF-κB inhibitor (BAY117082). Values were presented as mean ± SD (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed t-test).