Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2021 Oct 1.

Published in final edited form as: Biochem Pharmacol. 2020 Jul 4;180:114137. doi: 10.1016/j.bcp.2020.114137

Fig. 2. — The effect of LY3023414 on PI3K/AKT/mTOR signaling pathway and G0/G1 cell cycle arrest in human cancer cell lines. (A) Representative immunoblot images (upper panels) and quantification of integrated intensity (lower panels) of phosphorylation of AKT at T308 (pT308 AKT) or S473 (pS473 AKT), total Akt (t-AKT), phosphorylation of p70 ribosomal protein S6 kinase (pT389 p70S6K), p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6 phosphorylation (pS240/244 S6RP) and total S6 (t-S6), and tubulin of lysates from parental OVCAR-8 and multidrug-resistant NCI-ADR-RES cancer cells, as well as parental S1 and multidrug-resistant S1-M1-80 cancer cells treated with increasing concentrations (0 – 10 μM) of LY3023414 or 10 μM of a dual PI3K/mTOR inhibitor GDC-0980 (G). Representative immunoblot images (upper panels) and quantification of integrated intensity (lower panels) of pT308 AKT or pS473 AKT, t-Akt, ribosomal protein S6 phosphorylation (p-S6) and total S6 (t-S6), ABCB1, ABCG2 and tubulin of lysates from (B) OVCAR-8 and NCI-ADR-RES cells or (C) S1 and S1-M1-80 cells treated with DMSO (control), 2 μM of LY3023414 (+ LY) or 10 μM of a dual PI3K/mTOR inhibitor GDC-0980 (+ GDC) in the absence or presence of 1 μM of tariquidar or Ko143 in final DMSO concentration of 0.5% (v/v) as indicated for 2 h before processed for immunoblotting. Human EGF (50 ng/mL) was added to the culture medium for 5 min to stimulate phosphorylation. Quantification of relative levels of AKT phosphorylation in drug-sensitive (white bars) and multidrug-resistant (black bars) cells, presented as mean ± S.D. calculated from more than three independent experiments. (D) OVCAR-8 and NCI-ADR-RES cells, as well as S1 and S1-M1-80 cells, were plated and treated with DMSO (control), 2 μM of LY3023414, 1 μM of tariquidar or 1 μM of Ko143 alone or in combination as indicated for 24 h before harvest for cell cycle analysis. Quantifications of cells in each of the cell cycle stages are shown as mean ± S.D. calculated from more than three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, versus the same treatment in the absence of tariquidar or Ko143.

Fig. 2. — The effect of LY3023414 on PI3K/AKT/mTOR signaling pathway and G0/G1 cell cycle arrest in human cancer cell lines. (A) Representative immunoblot images (upper panels) and quantification of integrated intensity (lower panels) of phosphorylation of AKT at T308 (pT308 AKT) or S473 (pS473 AKT), total Akt (t-AKT), phosphorylation of p70 ribosomal protein S6 kinase (pT389 p70S6K), p70 ribosomal protein S6 kinase (p70S6K), ribosomal protein S6 phosphorylation (pS240/244 S6RP) and total S6 (t-S6), and tubulin of lysates from parental OVCAR-8 and multidrug-resistant NCI-ADR-RES cancer cells, as well as parental S1 and multidrug-resistant S1-M1-80 cancer cells treated with increasing concentrations (0 – 10 μM) of LY3023414 or 10 μM of a dual PI3K/mTOR inhibitor GDC-0980 (G). Representative immunoblot images (upper panels) and quantification of integrated intensity (lower panels) of pT308 AKT or pS473 AKT, t-Akt, ribosomal protein S6 phosphorylation (p-S6) and total S6 (t-S6), ABCB1, ABCG2 and tubulin of lysates from (B) OVCAR-8 and NCI-ADR-RES cells or (C) S1 and S1-M1-80 cells treated with DMSO (control), 2 μM of LY3023414 (+ LY) or 10 μM of a dual PI3K/mTOR inhibitor GDC-0980 (+ GDC) in the absence or presence of 1 μM of tariquidar or Ko143 in final DMSO concentration of 0.5% (v/v) as indicated for 2 h before processed for immunoblotting. Human EGF (50 ng/mL) was added to the culture medium for 5 min to stimulate phosphorylation. Quantification of relative levels of AKT phosphorylation in drug-sensitive (white bars) and multidrug-resistant (black bars) cells, presented as mean ± S.D. calculated from more than three independent experiments. (D) OVCAR-8 and NCI-ADR-RES cells, as well as S1 and S1-M1-80 cells, were plated and treated with DMSO (control), 2 μM of LY3023414, 1 μM of tariquidar or 1 μM of Ko143 alone or in combination as indicated for 24 h before harvest for cell cycle analysis. Quantifications of cells in each of the cell cycle stages are shown as mean ± S.D. calculated from more than three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001, versus the same treatment in the absence of tariquidar or Ko143.