Fig 1.
Cell survival curves of A) A549, B) A427, C) NCI-H23, D) NCI-H358, E) NCI-H1975, F) NCI-H1650 cells treated with increasing doses of A-674563 and MK-2206 for 72 hours. Cells were incubated with cell proliferation reagent WST-1 for 2 hours and absorbance was read at 450nm. Optical density was then normalized to a 1% DMSO control. The data is presented as the percentage of cell survival relative to the DMSO control ± SEM of three independent trials. Statistical significance was determined with multiple T-tests using the Holm-Sidak method without assuming a consistent SD and is represented by *p<0.05.
Fig 2.
A) Basal mRNA expression levels of A) Akt1, B) Akt2 and C) Akt3 in six NSCLC cell lines presented as the Mean ± SD with letters representing groups that are statistically different from one another such that (a) represents A549 cells, (b) A427 cells, (c) NCI-H23 cells, (d) NCI-H358 cells, (e) NCI-H1975 cells, and (f) NCI-H1650 cells. Samples were normalized to an Hprt housekeeping gene and statistical significance (p<0.05) was determined with one-way ANOVA and Tukey’s Multiple Comparison Tests. Results are based on 3 biological replicates and two technical replicates. D) Representative western blot of the basal protein levels of total AKT, phosphorylated AKT at serine 473 or threonine 308, total AKT1, AKT2 and AKT3 as well as the phosphorylated forms of AKT1 and AKT2 in six NSCLC cell lines. Phosphorylated AKT3 was not evaluated as no specific antibody for phosphorylated AKT3 is currently available. β-actin served as a loading control.
Fig 3.
Cell cycle analysis of A) A549, B) A427, C) NCI-H23, D) NCI-H358, E) NCI-H1975 and F) NCI-H1650 cells treated over a 24-hour period treated with A) 0.15μM, B) 0.03μM, C) 0.07μM, D) 0.36μM, E) 0.18μM and F) 0.70μM of both A-674563 and MK-2206. Data is presented as the Mean ± SEM with letters representing groups that are statistically different from one another such that (a) represents untreated cells, (b) DMSO treated cells, (c) MK-2206 treated cells, and (d) A-674563 treated cells. Statistical significance (p<0.05) was determined with two-way ANOVA and Tukey’s Multiple Comparison Tests. Results are based on 3 biological replicates.
Fig 4.
Representative western blot results of A) A549 and B) A427 cells after treatment with MK-2206 and A-674563 at the IC50 determined for the A-674563 inhibitor. Cells were pre-treated for 1 hour in serum-free media and protein was extracted after 30 minutes, 1 hour, and 2 hours of treatment in serum-containing media. Results are based on two biological replicates.
Fig 5.
Representative western blot results of A) A549 and B) A427 cells after treatment with MK-2206 and A-674563 at the IC50 determined for the A-674563 inhibitor. Cells were pre-treated for 1 hour in serum-free media and protein was extracted after 30 minutes, 1 hour, and 2 hours of treatment in serum-containing media. Results are based on two biological replicates.
Fig 6.
Cell survival curves of A) A549, B) A427, C) NCI-H23, D) NCI-H358, E) NCI-H1975, and F) NCI-H1650 cells treated with increasing doses of A-674563, PHA-84812, and H89 2HCl for 72 hours. Cells were incubated with cell proliferation reagent WST-1 for 2 hours and absorbance was read at 450nm. Optical density was then normalized to a 1% DMSO control. The data is presented as the percentage of cell survival relative to the DMSO control ± SEM of three independent trials. Statistical significance was determined with multiple T-tests using the Holm-Sidak method without assuming a consistent SD and is represented by *p<0.05.
Fig 7.
A) Basal protein levels of p-CDK2 and CDK2 across six NSCLC cell lines. Protein expression levels of CDK2 and p-CDK2 in B) A549 and B A427 cells after treatment with MK-2206 and A-674563 at the IC50 determined for A-674563. Cells were pre-treated for 1 hour in serum-free media and protein was extracted after 30 minutes, 1 hour, and 2 hours of treatment in serum-containing media. Results are based on two biological replicates.