Figure 3. Veratrine and its purified form VTD increase UBXN2A level in vitro and in vivo.

A. A cell-based screen was conducted in search of compounds that induce the expression of the UBXN2A gene. The 3.9 K base of DNA upstream from the UBXN2A gene on human chromosome 2, including endogenous promoters and necessary enhancers as well as untranslated exon 1, was cloned into MCS-mGL.1, a Gaussia luciferase vector, and transiently transfected into HCT-116 colon cancer cells. We used empty MCS-mGL.1 for background expression. This cell line was used to screen over 1800 FDA (Food and Drug Administration) approved drugs, synthetic compounds, and natural products. A glow luciferase activity assay was conducted in triplicate. We found 12 potential candidates in the initial screen, which were confirmed again by the luciferase assay. B–C. 40 μM Veratrine sulfate (an unpurified form of VTD) resulted in a ∼twofold increase in luciferase activity when compared to control. D. WB experiments showed incubation of HCT-116 with Veratrine for 24 hours leads to up-regulation of UBXN2A, while Veratrine has no effect on p47 (UBXN2C), another member of UBXD family. GAPDH was used as a loading control. Staurosporine, as an alkaloid, was used as a negative control. E. IP injection of Veratrine (0.125 mg/kg) to C57Bl/6N mice for 28 days showed a selective upregulation of UBXN2A in small intestine and colon tissues, but no changes were observed in the liver of the same animals. F. VTD, a naturally occurring plant alkaloid of steroidal structure, is a component of Veratrine mixture. G. HCT-116 cells were treated with VTD (20 and 40 μM) and cell lysates were subjected to WB. GAPDH was used as a loading control. VTD increases UBXN2A protein levels in a dose-dependent manner.