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. 2018 Sep 21;9(74):33896–33911. doi: 10.18632/oncotarget.26084

Figure 3. Mechanisms involved in CAI-induced Mcl-1 decrease.

Figure 3

(A) Cells were treated or not (DMSO) with 5 μM CAI for 24h (left panel) and 48h (right panel) for IGROV1-R10 and 48h (left panel) and 72h (right panel) for OVCAR3 and SKOV3. Mcl-1 mRNA expression was assessed by real time quantitative RT-PCR. GAPDH was used as a housekeeping reference gene for normalization. Histograms represent the relative mRNA expression in treated cells normalized to that of control cells. Data are expressed as mean ± SEM of three independent experiments. Statistical differences were analyzed with a Student t-test: NS=non-significant; (B) Ovarian carcinoma cell lines were treated or not (DMSO) with 5 μM CAI for 48h for IGROV1-R10 and 72h for OVCAR3 and SKOV3. One hundred nM bortezomib were added for the last 24h of treatment. Mcl-1 expression was monitored by western blot; (C) Cells were treated with increasing dose of CAI for 48h for IGROV1-R10 and 72h for OVCAR3 and SKOV3. The effect of CAI 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).