Table 1.
Primer sets used for RT-PCR on PSCs for isoforms A–D and K, and within the deleted area (Intra) and spanning the deleted area (Spanning).
Figure 1.
A. Nile red lipid staining at day 2. B. αSMA antibody staining at day 7. C. GFAP antibody staining at day 7. Secondary antibody was conjugated to Alexa 488 (green) and nuclear stain was DAPI (blue). Representative images of 3 independent experiments. Scale bars are 50 µm.
Figure 2.
Gene map of the P2X7 receptor isoforms.
The map is made according to the NCBI reference sequences. Exons are numbered and start/stop codon are marked. The map illustrates the potential mRNA isoforms that detected in mouse PSCs. These are marked in light grey for both WT and KO samples. Expected KO isoform A and K hybrids are also depicted, with the NeoR cassette inserted in white and the inserted early stop codon labeled.
Figure 3.
Expression of the P2X7 receptor in PSCs.
A. PCR results from PSCs isolated from WT and KO mice illustrate the presence of all isoforms A–D and K; mRNA transcripts are also present in KO. The area disrupted by homologous recombination (In) is, however, absent. Similarly, the area spanning the disrupted area (Sp) is missing. B. Immunostaining of PSCs with primary antibody for the extracellular domain of the P2X7 receptor. Scale bar is 10 µM. C. Western blots on whole cell lysate with antibody against an extracellular domain (ex-dom) and the C-terminal part of P2X7 (C-term) in reducing (r) and non-reducing conditions. The loading control was αSMA and this blot was exposed for shorter times. Representative of 3 independent experiments.
Figure 4.
Cell death of PSCs in the presence of 5 mM ATP.
A. Behavior of PSCs isolated from WT and KO was monitored for 10 hours; and pictures were taken at 2 min intervals. A. Representative phase contrast images of the PSCs at 0 hours, 5 hours and 10 hours. The top lane are WT cells and the bottom lane are KO cells. B. The bar graph summarizes the number of cells that died in control conditions or after addition of 5 mM ATP (n = 4–5 independent experiments). C. AnnexinV-FITC and propidium ioidide stain was added 5 hours after addition of 5 mM ATP to WT cells. D. Caspase 3/7 activation 5 hours after cells were stimulated with 5 mM ATP. E. BrdU incorporation measured 48 hours after the addition of 5 mM ATP. BrdU incorporations were normalized to 1% serum conditions. Significant difference (P<0.05) from respective controls (#) and between WT and KO (*) is indicated.
Figure 5.
Differences in numbers of isolated and proliferating PSC cells.
A. Number of isolated PSCs from pancreas of WT and KO mice (n = 10). PSCs were counted immediately after isolation in 5 separate areas of the dish. B. Difference in proliferation of WT and KO PSC following isolation and cell culture, as measured daily with Cell Counting Kit 8. (n = 3–5). Significant difference (P<0.05) between WT and KO (*) is indicated.
Figure 6.
Effect of endogenous ATP and serum on PSCs proliferation.
A. Comparison of the DNA incorporation in WT and KO PSCs with and without 5 U/ml apyrase. All samples contained 1% serum (n = 9–23). Y-axis shows the BrdU incorporation in relative light units. In the presence of apyrase, there was no significant difference between proliferation of WT and KO cells. Significant difference (P<0.05) from respective controls (#) and between WT and KO (*) is indicated. B. Effect of serum on DNA incorporation in WT PSCs. Significant difference (P<0.05) from 1% serum controls (#) and between 0% and 0% serum with 5 mM ATP (§) is indicated.
Figure 7.
ATP can stimulate life and death in PSCs.
A. Effect of ATP in growth medium with 1% serum on BrdU incorporation in WT PSCs (n = 6–23). B. Effect of selected ATP concentrations (µM) on BrdU incorporation in WT and KO PSCs both normalized to their respective 0 ATP controls (n = 7–23). BrdU incorporations were normalized to 1% serum conditions for the respective cell type. Significant difference (P<0.05) from respective controls (#) and between WT and KO (*) is indicated.
Figure 8.
Effect of BzATP, A438079 and az10606120 on proliferation of PSC.
A. The effect of BzATP on WT and KO PSCs (n = 6–7). All results were normalized to 1% serum controls that were set to 100%. The effect of: B. the P2X7 receptor A438079 inhibitor (10 µM); C. and negative allosteric modulator az10606120 (10 µM) on the basal proliferation response and in WT PSCs stimulated state with 100 µM ATP (n = 4–23). Significant difference (P<0.05) from respective controls (#) and with/without the inhibitor (*) is indicated.
Figure 9.
Calcium responses to BzATP and ATP in WT and KO PSC.
The Fluo-4 response (Ft/F0) of WT (A–D) and KO (E–H) PSCs stimulated with 50 µM BzATP, 50 µM ATP and lastly with 5 µM ionomycin. Intracellular Ca2+ signals were monitored in control conditions (A, E), in presence az101606120 (B, F), suramin (C, G) and suramin together with az10606120 (D, H). The figures illustrate one independent representative run with the frame average for about 50 cells in black, and an example response of 5 individual cells is shown in color. The peak intracellular Ca2+ responses to agonists +/− antagonists derived from frame intensities for approximately 50 cells/preparation were averaged for 4 separate PSCs preparations form WT and KO mice and shown as bargraphs. The letters underneath the bars show from which experiment (indicated by a letter) the mean Ca2+ peak values were taken from. Significant difference (P<0.05) from respective controls (#) and in between WT and KO preparations (*) is indicated.