FIGURE SUMMARY
Title

Antitumor Effect of Berberine Analogs in a Canine Mammary Tumor Cell Line and in Zebrafish Reporters via Wnt/β-Catenin and Hippo Pathways

Authors
Sammarco, A., Beffagna, G., Sacchetto, R., Vettori, A., Bonsembiante, F., Scarin, G., Gelain, M.E., Cavicchioli, L., Ferro, S., Geroni, C., Lombardi, P., Zappulli, V.
Source
Full text @ Biomedicines
Figure 1

Biochemical structure of berberine (BBR), berberrubine (BRR), and analogs.
Figure 2

Effect of berberine (BBR), berberrubine (BRR) and other analogs on CF33 cell viability. (a) Cells were treated with 2 μM, 5 μM, 10 μM, 20 μM, and 40 μM of the different compounds for 24 h. (b) Cells were treated with 10 μM of the different compounds and cell viability was measured at different time points (3, 6, 12, 15, 18, and 24 h). BBR, BRR and analogs exerted a dose- and time-dependent inhibition of cell viability.
Figure 3

Cell death after drug treatment. Early apoptosis and late apoptosis/necrosis rates of CF33 cells treated with 10 μM of the different compounds at 6 h, 12 h, 18 h, and 24 h, measured using flow cytometry with double staining for annexin V and propidium iodide (PI). Early apoptosis cells were Annexin V+/PI−, whereas late apoptosis/necrosis were Annexin V+/PI+. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4

Protein expression after drug treatment. Representative cropped Western blots of YAP/TAZ, β-catenin and active β-catenin in protein lysates obtained from CF33 cells treated with berberine (BBR), NAX035, NAX053, NAX057, NAX060 and berberrubine (BRR) for 24 h. Bands of the expected sizes for YAP (~70 kDa), TAZ (~55 kDa), β-catenin (~92 kDa) and active β-catenin (~92 kDa) are present in all samples. The experiment was performed in quadruplicate. Each lane corresponds to a replicate. Ponceau red was used as a loading control.
Figure 5

mRNA expression after drug treatment. Gene expression analysis by semi-quantitative PCR of genes directly or indirectly involved in Wnt/β-catenin and Hippo pathways in CF33 cells treated with berberine (BBR), NAX035, NAX053, NAX057, NAX060 and berberrubine (BRR). (a) β-catenin, (b) YAP, (c) TAZ, and (d) CTGF mRNA expression levels in CF33 cells. Relative gene expression levels are shown following normalization with beta-actin. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 6

Wnt/β-catenin and Hippo pathways zebrafish reporters. Tissue specificity expression of mCherry under control of (a) TCF/LEF and of (b) CTGF genes. Quantification of mCherry fluorescence in control transgenic zebrafish in comparison with transgenic zebrafish treated with berberine (BBR) and other analogs (NAX035, NAX053, NAX057). *, p < 0.05; ***, p < 0.001.
Acknowledgments
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