Figure 3. P2RX4 is required for CAD-induced Ca2+ and cAMP responses and cell death.
(A) Representative confocal images of MCF7 cells transfected with P2RX4-pHluorin [29] and LAMP1-mCherry for 72 h. Scale bar, 10 μm.
(B) Representative immunoblots of P2RX4 and β-actin (loading control) in lysates of MCF7 cells treated with non-targeting control (NTC) or P2RX4 siRNAs for 72 h.
(C) Fluo-4-AM geometric mean fluorescence intensities (geo-MFIs) in MCF7 cells treated with indicated siRNAs for 72 h and with 6 μM siramesine in the absence of extracellular Ca2+ for the last 10 min. The values are means of actual geo-MFIs (left) or geo-MFIs relative to untreated samples (right).
(D) Cell death of MCF7 cells treated with indicated siRNAs for 72 h and with indicated concentrations of siramesine for the last 48 h.
(E) Fluo-4-AM geo-MFIs in MCF7 cells treated with indicated siRNAs for 72 h and with 300 μM GPN in the absence of extracellular Ca2+ for the last 8 min. The values are means of geo-MFIs relative to untreated samples.
(F) Flamindo2 geo-MFIs in MCF7-Flamindo2 cells treated with indicated siRNAs for 72 h and with 6 μM siramesine in the absence of extracellular Ca2+ for the last 15 min. The values are presented as means of actual geo-MFIs (left) or geo-MFIs relative to untreated samples (right).
(G) Normalized geometric MFIs in MCF7-Flamindo2 cells treated with DMSO (control), 10 μM BAPTA-AM, 20 μM ML-SA1, 2 mM LLOMe, 200 μM GPN or 5 μM forskolin in the absence of extracellular Ca2+ for 20 min.
(H) Representative immunoblots of P-S133-CREB and lamin B (loading control) in lysates of MCF7 cells treated with 2 mM LLOMe or 6 μM siramesine for indicated times.
Error bars, SD of ≥ 3 independent experiments. * P < 0.05, ** P < 0.01 as analyzed by 2-tailed, homoscedastic student’s t-test.