Fig. 9.

Drug response of ALI multilayered co-cultures in the presence of mTOR inhibitor rapamycin. a Brief schematics of the PI3K/AKT/mTOR and MEK/ERK signalling cascades and the inhibitory effect of rapamycin. b Western blot analysis of the expression of phospho-mTOR (p-mTOR), cIAP-1/2 and phospho-p42/44 (ERK1/2) (p-p42/44) in ALI multilayered co-cultures grown for 72 h and exposed to rapamycin (72 h) by direct inoculation. Three concentrations of rapamycin were tested: 0.1, 1 and 100 μM. Untreated cultures (−) were also analysed for comparison. α-tubulin expression is reported as protein loading control. Histogram of the LDH activity in the experimental controls are also reported: untreated ALI multilayered co-cultures (NT), ALI multilayered co-cultures exposed by direct inoculation to rapamycin (0.1, 1 and 100 μM) for 72 h, and positive control (LDH PT). Significant LDH activity was detected following 100 μM rapamycin treatment. Data are reported as average ± standard error of the mean (n_replicates = 2; n_tests = 3). p < 0.01 and p < 0.001 indicate a significant difference from NT (one-way ANOVA with Dunnett post-test). c 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 rapamycin (0.1, 1 and 100 μM). Cell cultures were exposed 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 (n_replicates = 2; n_tests = 3). No significant differences from NT were found (two-way ANOVA with Bonferroni post-test)