Fig 2: Examining the influence of P2RX inhibitors in combination with E-NTPDase inhibitor on cell viability and eATP release in paclitaxel-treated cells. (A) Paclitaxel-treated breast cancer MDA-MB 468 cell lines were treated for six hours with P2RX7 inhibitor A438079 (20 µmol/L) or P2RX4 inhibitor 5-BDBD (20 µmol/L) in the presence or absence of PSB 069 (10 µmol/L), and cell viability was measured by applying PrestoBlue HS assay. Standard deviation was calculated from three independent experiments performed in triplicate. We used the same values for both graphs for vehicle addition and PSB 069. (B) eATP concentrations were measured in the supernatants of paclitaxel-treated MDA-MB 468 cells after six hours of treatment. Standard deviation was calculated from three independent experiments performed in triplicate. We used the to the same values for both graphs for vehicle addition and PSB 069. The student’s t-test was applied to the applicable assays to ascertain significance. * represents p < 0.05 and ** represents p < 0.01 for A438079 and PSB 069 or 5-BDBD and PSB 069 when compared to PSB 069 alone.

Fig 3: mRNA and protein expression analysis of P2RX4 and P2RX7 for all cell lines. (A) qRT-PCR was performed on mRNA of TNBC cell lines and MCF-10A cells using specific primers for P2RX4 and P2RX7. * represents p < 0.05 and ** represents p < 0.01. TNBC cell lines, MCF-10A cells, and HEK 293T cells transfected with P2RX4 or P2RX7 as positive controls were probed for (B) P2RX4 and (C) P2RX7, and GADPH was used as a loading control for western blot analysis repeated twice. HEK 293T cells transfected with P2RX7 were loaded at increasing protein concentrations of 1.0 µg, 2.5 µg, and 5.0 µg combined with lysates of control vector-transfected cells to keep the total loaded protein the same in each lane. Densitometry analysis was performed using Image Studio on the 75 kDa P2RX7 band. The student’s t-test was applied to the applicable assays to ascertain significance. * represents p<0.05 and ** represents p < 0.01 relative to MCF-10A; + represents p < 0.05 and ++ represents p < 0.01 relative to HEK293-empty vector transfected. (D) The calculated difference in mean fluorescence intensity (MFI) values between TNBC cell lines, MCF-10A cells, and HEK 293T cells transfected with P2RX4 or P2RX7 as positive controls stained with P2RX4 or P2RX7 specific antibody and the isotype control for the different cell lines examined. * represents p < 0.05 and ** represents p < 0.01 relative to MFI difference in MCF-10A cells; + represents p < 0.05 and ++ represents p < 0.01 relative to MFI difference in HEK293-empty vector transfected. O/E represents overexpressed.

Fig 4: Determining relative eATP content and cell viability in paclitaxel-treated cells in the presence of ivermectin or vehicle addition. (A) The graphs represent cell viability as measured using the Presto Blue HS assay +/- standard deviation from three independent experiments performed in triplicate in TNBC and MCF-10A cells after six hours of treatment with increasing concentrations of paclitaxel and the P2RX4 and P2RX7 activator ivermectin (10 µmol/L) or vehicle addition. (B) eATP content was measured in the supernatants of paclitaxel-treated TNBC and MCF-10A cell lines in the presence of the P2RX4 and P2RX7 activator ivermectin (10 µmol/L) or vehicle addition. The student’s t-test was applied to the applicable assays to ascertain significance. * represents p < 0.05 and ** represents p < 0.01 when comparing ivermectin to vehicle addition.

Fig 5: Schematic displaying our proposed model for ATP release. Our proposed model suggests that ivermectin activates P2RX4 and P2RX7 leading to the release of ATP and the more ATP that accumulates extracellular can promote cell death especially in the presence of paclitaxel. In addition, the breakdown of ATP can be prevented in the presence of E-NTPDase inhibitors POM-1 or PSB 069. However, the release of ATP can be prevented in the presence of P2RX4 inhibitors 5-BDBD or Iso-PPADS or P2RX7 inhibitors A438079 or Iso-PPADS.