Figure 4. ADCY1 depletion inhibits CAD-induced cAMP response and cell death.

(A) Cell death of MCF7 cells treated with non-targeting control (NTC) and 3 independent ACDY1 siRNAs for 72 h and with indicated concentrations of siramesine for the last 48 h (left). The efficacy of siRNAs was analyzed by qPCR 48 h after the transfection (right).

(B) Representative flow cytometer profiles for Flaminod2 fluorescence in MCF7-Flamindo2 cells treated with NTC or ADCY1 #1 siRNAs for 48 h. When indicated, cells were treated with vehicle (DMSO) or 6 μM siramesine in the absence of extracellular Ca²⁺ for the last 20 min.

(C) Relative Flamindo2 geometric mean fluorescence intensities (geo-MFIs) in MCF7-Flamindo2 cells treated with NTC or ADCY1 #1 siRNAs for 72 h.

(D) Kinetics of relative Flamindo2 geo-MFIs in MCF7-Flamindo2 cells transfected as in (C) and treated with 6 μM siramesine in the absence of extracellular Ca²⁺ for the last 25 min.

(E and F) Representative immunoblots of P-S133-CREB and HSP90 (loading control) in MCF7 cells transfected with siRNAs as in (C) and treated with indicated concentrations of CADs for the last 2 h (E), and mean P-S133-CREB/loading control ratios in indicated samples from 3 independent experiments (F)

(G) Levels of free [Ca²⁺]_c in MCF7 cells treated with NTC or ADCY1 #1 siRNAs for 72 h, and with 6 μM siramesine (left) or with 300 μM GPN (right) in the absence of extracellular Ca²⁺ for indicated times were analyzed by flow cytometry using Fluo-4-AM probe as a Ca²⁺ sensor.

Error bars, SD of ≥ 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as analyzed by 2-tailed, homoscedastic student’s t-test.