Fig 2: Cell viability of ATP-treated cells in the presence of P2RX inhibitors. TNBC cell lines and MCF-10A cells were treated for 48 hours with increasing concentrations of ATP in the presence of the P2RX inhibitor Iso-PPADS (20 µmol/L), the P2RX7 inhibitor A438079 (20 µmol/L) or the P2RX4 inhibitor 5-BDBD (20 µmol/L) or vehicle addition, and cell viability was measured using the PrestoBlue HS assay. Error bars represent standard deviations calculated from three independent experiments performed in triplicate. One way ANOVA with Tukey’s HSD was applied to ascertain significance. * represents p < 0.05 and ** represents p < 0.01 when comparing vehicle addition to Iso-PPADS, A438079 or 5-BDBD.

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.