Fig. 8
Drug response of ALI multilayered co-cultures in the presence of perifosine, an inhibitor of phospho-AKT. a Simplified schematics of the PI3K/AKT/mTOR signalling cascade and perifosine inhibitory effect. b Western blot analysis of the expression of AKT and phospho-AKT (p-AKT) in ALI multilayered co-cultures grown for 72 h and exposed to perifosine (2.5 μM; 72 h) by direct inoculation. Untreated cultures (−) were also analysed as controls. GADPH expression was the protein loading control. c Histogram of the LDH activity in the experimental controls: untreated ALI multilayered co-cultures (NT), ALI multilayered co-cultures exposed by direct inoculation to perifosine (2.5 μM) for 72 h, and positive control (LDH PT). No significant increase in LDH activity was detected following perifosine treatment as compared to NT. Data are reported as average ± standard error of the mean (nreplicates = 2; ntests = 3). p < 0.001 indicates a significant difference from NT (one-way ANOVA with Dunnett post-test). d Percentage (%) cytotoxicity detected by LDH cytotoxicity assay in ALI multilayered co-cultures grown for 72 h and exposed to 10 concentrations of docetaxel for 72 h, in the presence or absence of perifosine (2.5 μM). Cell cultures were exposed to drugs by direct inoculation. Values for untreated cultures (NT) and positive control (LDH PT) are also shown. Data are reported as average ± standard error of the mean (nreplicates = 2; ntests = 3). p < 0.001 indicates a significant difference from NT (two-way ANOVA with Bonferroni post-test)