Pgl4.42 luc2p hre hygro vector

Luciferase assays were performed as described previously^{74 (link)}. In accordance with the manufacturer’s instructions, Cignal Reporter Assay Kit STAT3 (QIAGEN, Hilden, Germany) was used as a STAT3 reporter plasmid. The HIF-1 reporter plasmid [pGL4.42 (luc2P/HRE/Hygro) vector] was purchased from Promega (Madison, WI, USA), and the NF-κB reporter plasmid used in this study was described^{73 (link)}. Forty-eight hours after transfection, reporter activity was assessed by GLOWMAX (Promega) with the Dual-luciferase reporter assay system (Promega).

All obtained transduced variants of 1BR.3.N cells were cotransfected using FUGENE HD transfection reagent (Promega Corporation, Madison, WI, USA) with 1 μg of pGL4.42 [luc2P/HRE/Hygro] vector (Promega Corporation, Madison, WI, USA) together with 0.1 μg of pGL4.75 Renilla luciferase reporter vector (Promega Corporation, Madison, WI, USA), used as a normalization control. Transfection was performed according to the manufacturer’s instruction. The cells were afterwards cultured at standard condition (5% CO₂; 37 °C) for 24 h. Then, the transfection’s medium were removed and 75 µL of the medium was added: to the first one 5mM glucose concentration and 25 mM to the other. One of the plates was then incubated for 6h under hypoxia conditions (1% O₂) and the next under normoxic conditions (19% O₂). After incubation, the luciferase activity in each group was measured using the Dual-Glo Luciferase Assay System (Promega Corporation, Madison, WI, USA) by the VICTOR Multilabel plate reader (PerkinElmer, Waltham, MA, USA).

To explore the potential ASCL1 binding sites in the IGFBP5 gene, fragments of IGFBP5 that are upstream and downstream of the genomic transcription start site were amplified and subcloned into the pGL4.42 (luc2P/HRE/Hygro) vector (Promega) using the XhoI and KpnI sites. Ten reporter vectors were generated with this strategy, pGL4.P1 (P1, −1.5 kb ∼−50 bp), pGL4.P2 (P2, −0.6 kb ∼−50 bp), pGL4.P3 (P3, +0.1 kb ∼+0.9 kb), pGL4.P4 (P4, +2.9 kb ∼+3.2 kb), pGL4.P5 (P5, +3.2 kb ~+3.5 kb), pGL4.P6 (P6, +6 kb ~+6.6 kb), pGL4.P7 (P7, +6.6 kb ~+7 kb), pGL4.P8 (P8, +7.2 kb ~+7.9 kb), pGL4.P9 (P9, +7.9 kb ~+8.4 kb) and pGL4.P10 (P10, +9 kb ~+10 kb). The E-box-deficient pGL4.42 mutants reporters were generated using the QuikChange site-directed mutagenesis kit (Stratagene). The sequences of the primers are listed in Supplementary Table 3. To perform the luciferase reporter assay, these pGL4 reporter vectors were co-transfected with pLenti-V5-ASCL1, pLenti-V5-NEUROD1 or pLenti-V5-GFP into HEK293T cells. The pGL4–hRluc expressing the Renilla reniformis luciferase under the TK promoter was used as the internal control in each experiment. Forty eight hours after transfection, cells lysates were collected and were used to determine the luciferase activity using the protocol from Promega. Experiments were performed in triplicate.