Fig 2: Expression of P2X7R and cell proliferation with ATP, AZ10606120 and BzATP.(a) Representative gel of P2X7 mRNA expression (284 bp) in HPDE (H) and PancTu-1 Luc (P) cells (n = 3). (b) Western blot on the whole cell lysates with polyclonal C-terminal antibody for P2X7R shows the A isoform (70 kDa). ß actin (42 kDa) was used as loading control (n = 4). Bar graph shows the level of P2X7R protein as a ratio to ß actin. Significant difference in comparison to HPDE cells p < 0.001 (**) is reported. (c) Effect of ATP and ATP plus AZ10606120 on PancTu-1 Luc cell proliferation was assayed using BrdU incorporation. Black bars show the effect of increasing concentration of exogenous ATP (0 µM, 10 µM, 100 µM and 1 mM). White bars represent the effect of increasing ATP concentration in combination with 10 µM AZ10606120 (P2X7R allosteric inhibitor). (d) Effect of increasing concentrations of BzATP (P2X7R agonist) on BrdU incorporation. Each run was performed in triplicates and the graphs show data from four independent experiments (mean ± SEM). Significant differences p < 0.05 (*, #) and p < 0.001 (**, ##) from the respective control, without exogenous ATP or BzATP (*, **) and with/without inhibitor (#, ##) are indicated. (e) The graph shows PancTu-1 Luc cell proliferation after transfection with siRNA against P2X7 mRNA (siP2X7), scramble siRNA (siSCR) and siP2X7 plus BzATP (100 µM). Data are represented as mean ± SEM. Significant differences p < 0.001 (**) from the control are indicated. (f) Western blot shows the protein expression of P2X7R in PancTu-1 Luc cells transfected with siRNA against P2X7R (siP2X7) or scramble Naito-1 siRNA as control (siSCR).

Fig 3: P2X7R expression in PancTu-1 Luc in vivo model.(a) Immunofluorescence detection of EGFR in the tumour cells (red), a-SMA for the pancreatic stellate cells (cyan) and P2X7R (ATTO-488, green). The staining was performed in two subsequent 2 µm sections. (b–d) Representative pictures of P2X7R expression in PancTu-1 Luc tumour paraffin sections. The slides were stained with three different P2X7R antibodies: (b) APR-008 against an epitope in the extracellular loop. (c) APR-004 against the C-terminal; (d) PA5-28020 against the C-terminal and more specific for human tissues embedded in paraffin. (e) Control of P2X7R antibody (PA5-28020) in a tumour-free pancreas of the PDAC mice. In images (b–d) following structures are indicated: Islets of Langerhans (a), ducts (b), PanINs (c) and acinar-like structures with increased lumen size (arrows). All images are representative of staining carried out on n = 3–6. Images were taken with different magnifications but scale bars are 50 µm on all panels. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Fig 4: P2X7 in DCs favor the membrane transfer from ACs to MutuDC cells(A and B) CellMask transference from H-ACs to the surface of the BM-DCs (A) and P2X7-KO BM-DCs (B) was analyzed to assess the role of DC P2X7 in membrane transference. Graphs show the fluorescence intensity on the surface of BM-DCs associated with ACs (red) and BM-DCs not associated with ACs (control, open black).(C) Graph shows the slope derived from each fluorescence intensity curve.(D) P2X7 expressing or parental HEK293 cells were loaded with CellTracker Orange (CMRA), and H-ACs were obtained by nutrient deprivation. The orthogonal projection of H-ACs shows the whole-cell distribution of fluorescence. ACs are shown as white pseudocolor.(E and F) Representative histograms and (F) percentage of BM-DCs which have phagocytosed CMRA labeled ACs. H-ACs and H-ACs-P2X7 phagocytosed by CD11c+ 7-AAD+ WT DCs (Upper panel) or CD11c+ 7-AAD+ P2X7-KO DCs (Lower panel). Phagocytosis of fluorescence latex beads was the positive control. The quantification of three independent experiments was graphed and represented as the mean ± SEM. Results were analyzed using non-parametric Mann-Whitney test, * represent significant differences p < 0.05 and ** p < 0.01, treatments compared against control, n/s; not significant.

Fig 5: Effect of AZ10606120, BzATP and mPSCs on PancTu-1 Luc cells migration.PancTu-1 Luc cells were grown to confluence in the two-chamber silicon insert. After removal, phase-contrast images were taken every hour for 30 hr. (a) Representative images at 0, 15 and 30 hr for control, AZ10606120, BzATP and P2X7R inhibitor plus BzATP. (b) White, black, grey and light grey circles show the percentage of the wound/gap open every hour in control cells (vehicle); cells treated with 10 µM AZ10606120, with 10 µM BzATP and AZ10606120 plus BzATP, respectively. Significant differences p < 0.05 (*) between control and inhibitor/agonist was calculated from the slope of the curves after 11 hr. (c) The graph shows the endpoint, i.e., percentage of wound still open after 30 hr; significant differences p < 0.05 (*) and p = 0.001 (**) are indicated. Four to seven fields of view were analyzed per each experiment. The graphs show data from 3 to 4 independent experiments (mean ± SEM). (d) The graph shows effect of mPSCs from wild-type and P2X7 knockout (+/-AZ10606120) on PancTu-1 Luc cells migration. The graph shows data from four independent experiments (mean ± SEM). Significant differences p < 0.001 (**) are indicated.