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. 2020 Jun 23;11:411. doi: 10.3389/fendo.2020.00411

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Copyright © 2020 Ropolo, Catrinacio, Renna, Boggio, Orquera, Gonzalez and Vaccaro.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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VMP1 promoter analysis. (A) To assay VMP1 promoter activity, a 3,005 bp sequence of the 5′ upstream region of the VMP1 gene was amplified and cloned into the pGL3 reporter vector (pGL3.vmp1-3005) driving Luciferase (Luc) expression. (B) qPCR assay showed an increase of VMP1 mRNA expression in HeLa and PANC-1 cells submitted to starving conditions and rapamycin treatment for 4 h (n = 3). Next, HeLa and PANC-1 cells were transfected with pGL3.vmp1-3005. (C) Relative Luciferase activity shows an increase of VMP1 promoter activation in HeLa and PANC-1 cells submitted to starving (Stv) conditions and rapamycin (Rapa) treatment. (n = 3). (D) Relative Luciferase activity shows an increase of VMP1 promoter activation in HeLa and PANC-1 cells submitted to gemcitabine treatment for 24 h (n = 3). (E) Consecutive shorter fragments of 3005 bp sequence of VMP1 promoter were amplified and subcloned in the pGL3 reporter vector (pGL3.vmp1-1977, pGL3.vmp1-1469, pGL3.vmp1-883). (F) HeLa cells were submitted to starvation conditions, rapamycin and gemcitabine treatment for 24 h. Relative Luciferase activity showed higher levels of VMP1 promoter activation in the shorter construct (pGL3.vmp1-883) compared to 3,005 bp sequence of VMP1 promoter in all treatments. *p < 0.05.