Pgl4.13 vector

Six human glioma cell lines (U251, U118, U373, A172, U87MG, and T98G) were maintained in DMEM containing 10% FBS, 4 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Short tandem repeat (STR)–PCR profiling demonstrated that the U373MG (Sigma Catalogue number 89081403) and U251 (ECAC catalog 09063001) cells originated from a common cell line [71 (link)]; however, these cells showed different biological properties and are models of different evolutionary strains of the same original tumor cell line. It is well known that in vitro subcultures represent, indeed, a selection pressure on cell lines, and, over time, this may result in genetic drift in the cancer cells. Luciferase-transfected U87MG cells were kindly provided by J.E. Heikkila (Abo Akademi University, Turku, Finland). Three patient-derived glioma stem cell lines (BT12M, BT48EF, and BT50EF) were kindly provided by J.G. Cairncross and S. Weiss (University of Calgary, Canada) [72 (link)], and one (CSCs-5 [73 (link)]) was provided by M. Izquierdo (Universidad Autónoma de Madrid, Spain). Luciferase was inserted into the genome of GSC-5 cells using the pGL4.13 vector (Promega, Milan, Italy) and the jetPEI DNA transfection method (Polyplus, Illkirch, France).

The wide type ZEB1 3'-untranslated region (UTR) containing miR-369 targeting sequence (GUAUUAUU) and the mutated type (AGCGAGUU) was amplified and cloned into the luciferase reporter plasmid pGL4.13 vector (Promega, Madison, WI). All the constructs were verified by sequencing. Briefly, laryngocarcinoma cells were co-transfected with miR-369 mimic or miR-control and pMIR-reporter luciferase vector containing a specific sequence of wild-type or mutant ZEB1 fragment, using siRNA transfection (Invitrogen, NY, USA). Cells were collected and lysed for luciferase detection 48 hours after transfection. The relative luciferase activity was normalized against to the Renilla luciferase activity.

Twelve human glioma cell lines (U251, U373, U118, U138, A172, U87MG, SW1783, SNB19, LN229, T98G, SF268, and D54) were purchased from ATCC or DSMZ cell collections and were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% FBS, 4 mM glutamine, 100 IU/mL penicillin, and 100 μg/mL streptomycin. To minimize the risk of working with misidentified and/or contaminated cell lines, cells were stocked at very low passages. However, periodically, DNA profiling was carried out by using the AmpFLSTRTM identifillerTM PCR amplification kit from Thermo Fisher following the manufacturer’s instructions. Luciferase-transfected U87MG cells were kindly provided by J.E. Heikkila (Abo Akademi University, Turku, Finland). Three GBM patient-derived stem cell lines (BT12M, BT48EF, BT50EF) were kindly provided by J.G. Cairncross and S. Weiss (Arnie Charbonneau Cancer Institute, University of Calgary, Canada) [45 (link),46 (link)], and GSCs-5 and GSCs-7 [47 (link),48 (link)] were from M. Izquierdo (Universidad Autónoma de Madrid, Spain). Luciferase was inserted in the genome of GSCs-5 cells using the pGL4.13 vector (Promega, Milan, Italy) and the jetPEI DNA transfection method (Polyplus, Illkirch, France). Molecular characteristics of the 12 cell lines used in this report are summarized in Supplementary Table S1.

The longest 5′-UTRs of human Vegfr-1/2/3 and Egfr annotated in RefSeq (NCBI Reference Sequence Database; www.ncbi.nlm.nih.gov/RefSeq/) were cloned into the pGL4.13 vector (Promega) between the SV40 promoter and the Firefly luciferase open reading frame using HindIII (New England Biolabs, Ipswich, MA) and AvrII (New England Biolabs). Deletion mutants of a putative internal ribosome entry site (IRES) between -87 and -1 of the Vegfr-1 5′-UTR and an authentic IRES (core region: −56 to −20) between −56 and −1 of Egfr were also subcloned into the same cloning sites of the pGL4.13 vector. IRES sequences were identified via computational prediction using the IRESpy program (https://irespy.shinyapps.io/IRESpy/). HEK-293 cells were cotransfected with a pGL4.13/5′-UTR reporter construct and Renilla luciferase pGL4.74 vector as a normalization control reporter using Lipofectamine 3000 (Invitrogen), followed by treatment with or without DOX or infection with or without Ad-Redd1. Cell lysates were prepared 24 h later, and luciferase activity was measured using the Dual Luciferase Assay Kit (Promega). Firefly luciferase activity was normalized to Renilla activity.