A1207 and U87 cells (2 × 104 per well) were seeded in a 24-well plate. After 24 h, the cells were transfected (HiPerfect, QIAGEN, Hilden, Germany) with 50 nM hsa-miR-29b-3p-5-FAM-labeled (miR-29b-mimic) or with 100 nM hsa-miR-29b-3p inhibitor (miR-29b-inh) (QIAGEN, Hilden, Germany) for 48 h. These concentrations have already been used in other studies.72 (link),73 ,74 (link) Cells transfected with scrambled-mimic-5-FAM-labeled or scrambled-inhibitor (QIAGEN, Hilden, Germany) served as controls (ctrl), respectively.
For the overexpression of Sp1,75 (link) H19,76 (link) and c-Myc, (addgene; Watertown, MA, USA) A1207 and U87 cells (4 × 104 per well) were seeded in a 24-well plate. After 24 h, the cells were transfected (Lipofectamin 3000; Thermo Fisher Scientific, Damstadt, Germany) with 250 ng pEF_Sp1, pcDNA_H19, pcDNA_c-Myc or the corresponding control plasmids (ctrl) for 48 h.
For target validation, HEK293 cells were transfected (Lipofectamin 3000) with pGL4, pGL4_NG2-promoter,19 (link) pGL4_WT-NG2-3UTR, pGL4_MUT-NG2-3UTR, pGL4_WT-PDGFRA-3UTR, or pGL4_MUT-PDGFRA-3UTR (250 ng) and co-transfected with the miR-29b-mimic (50 nM) and pEF_Sp1, pcDNA_H19 or pcDNA_c-Myc (250 ng).
For the overexpression of Sp1,75 (link) H19,76 (link) and c-Myc, (addgene; Watertown, MA, USA) A1207 and U87 cells (4 × 104 per well) were seeded in a 24-well plate. After 24 h, the cells were transfected (Lipofectamin 3000; Thermo Fisher Scientific, Damstadt, Germany) with 250 ng pEF_Sp1, pcDNA_H19, pcDNA_c-Myc or the corresponding control plasmids (ctrl) for 48 h.
For target validation, HEK293 cells were transfected (Lipofectamin 3000) with pGL4, pGL4_NG2-promoter,19 (link) pGL4_WT-NG2-3UTR, pGL4_MUT-NG2-3UTR, pGL4_WT-PDGFRA-3UTR, or pGL4_MUT-PDGFRA-3UTR (250 ng) and co-transfected with the miR-29b-mimic (50 nM) and pEF_Sp1, pcDNA_H19 or pcDNA_c-Myc (250 ng).
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