Fig. 3.

HO53 activates the AMPK signaling cascade in differentiated BCi cells. Differentiated BCi cells in the ALI culture were treated with HO53 (75 μM) for 4 h, 8 h, and 24 h. aHeatmap showing changes in expression as log2FC of genes related to the mTOR/AMPK pathway at 4 h, 8 h, and 24 h of treatment with HO53. The heatmap shows significantly differentially expressed genes in comparison to control cells collected at the same time points (p < 0.05). Genes selected for further analysis were marked in red. Nonsignificant differentially expressed genes have arbitrary value 0. Expression level of LGALS9B/C (b) and PRKAA2 (c) genes analyzed by qRT-PCR. Gene expression was represented as a fold change in comparison to control cells (value 1, dashed line) collected at the same time point as treated cells and normalized to the EEF2 reference gene. n = 3 trans-well inserts from 3 independent experiments ± SEM. *p < 0.05, ***p < 0.001 versus control cells analyzed by one-way ANOVA with the Dunnett post hoc test. dActivation of the AMPK signaling pathway analyzed by Western blotting of phosphorylated AMPKα (Thr172) at different time points of HO53 treatment. Quantification of Western blots was performed in two steps: first, by measurements of band intensity and normalization to GAPDH (loading control) and then by calculation of the normalized ratio for phospho-AMPKα (Thr172) to total AMPKα. The representative Western blot of n = 4 independent experiments. Quantitative comparison was done on the samples run in one experiment on separate gels/blots processed in parallel. Statistical analysis was performed by one-way ANOVA with the Sidak post hoc test, where *p < 0.05 versus corresponding solvent control. Full-length blots are presented in online supplementary Figure S7. log2FC, log₂ fold change; SEM, standard error of mean.