Figure 4. YM48583 blocks SOCE and inhibits mTORC1 targets and Mcl-1.

(A) Effect of YM58483 on IP3R and SOC. Microspectrofluorimetry using Fura-2AM probe was performed in the three carcinoma cell lines IGROV1-R10, OVCAR3 and SKOV3 cells. During exposure to 0Ca²⁺, depletion of the intracellular stores was triggered by the addition of 2 μM thapsigargin to the bathing medium. Subsequent replenishment of 2 mM Ca²⁺ to the medium elicited a rise in [Ca²⁺]_i due to Ca²⁺ influx through open store-operated channels. Black tracings depict the representative changes in [Ca²⁺]_i recorded from DMSO treated cells and grey tracings depict the representative changes in [Ca²⁺]_i recorded from cells pre-treated 1h with 15 μM for IGROV1-R10 and OVCAR3 cells or 20 μM for SKOV3 cells (data are representative of three independent experiments). (B) Effect of YM58483 on Akt/mTORC1 pathway. Cells were treated with YM58483 (15 μM for 48h for IGROV1-R10 cells, 15 μM for 72h for OVCAR3 cells and 20 μM for 72h for SKOV3 cells). The effect of YM58483 treatment on the activation of the PI3K/Akt/mTOR pathway was analyzed by studying the protein expression of P-Akt (Ser473 and Thr308) and total Akt, P-mTORC1 (Ser2448) and P-mTORC2 (Ser2448), P-p70S6K (Thr389) and total p70S6K and P-4E-BP1 (Thr70) and total 4E-BP1 by western blot analysis. Protein levels (standardized based on actin) were determined by densitometry scanning with Image J software to generate the values shown in the bar graphs. Results are expressed as mean ± SEM. Statistical differences were analyzed with a Student t-test: ^*p< 0.05, ^**p< 0.01, ^***p< 0.001 (n=3).