Fig. 2
Effect of CRE in different cis-acting elements of the GDNF promoter II on GDNF transcription. a U251 cells in the logarithmic growth phase were seeded into 24-well plates and then transfected with 0.8 μg each of wild-type (CRE-WT), deletion (△CRE-E, △CRE-S, and △CRE-ES), or mutant plasmids (mtCRE-E, mtCRE-S, and mtCRE-ES) of the GDNF promoter II and 80 ng of the internal reference plasmid (pRL-TK) when the cells reached 80% confluence. Luciferase activity was assessed 48 h after transfection. Firefly luciferase activity was normalized to Renilla luciferase activity. The letter E indicates enhancer II, while the letter S indicates silencer II (n = 3). b U251 cells infected with lentivirus (CREB-OE or EGFP-NC) were treated as described in (a), GDNF promoter II activity was assessed by dual-luciferase assay (n = 3). ** indicates P < 0.01 vs. pCRE-WT plus EGFP group; ## indicates P < 0.01 vs. EGFP-NC group with each type of promoter II; Δ∆ indicates P < 0.01 vs. wild-type GDNF promoter II overexpressing CREB. c sgRNA sites within the human GDNF promoter II. Thick arrows indicate the primer positions. Thin arrows indicate major TSSs. The letter E indicates enhancer II, while the letter S indicates silencer II. d After 72 h of infection with viral sgRNAs, the ability of the sgRNAs to direct Cas9 to cleave CRE in enhancer II and silencer II was assessed by CruiserTM assay. Black arrows indicate two DNA fragments formed by digestion. eGDNF transcription in Cas9-U251 cells was determined by real-time PCR 72 h after infection with sgRNA-CRE-E or sgRNA-CRE-S (n = 3). All data are mean ± SD. *P < 0.05; **, ##, and different uppercase letters indicate P < 0.01