Figure 2. USF1 was involved in GAS6-AS2 aberrant expression. (A) Intersection of “PROMO” and “JASPAR” algorithms predicted binding transcriptional factors (TFs) in GAS6-AS2 promoter. (B) Real-time PCR detected the expression of TFs in randomly selected three paired OS tumor specimens and normal tissues. (C) qPCR determined the levels of USF1, YY1, STAT3 and USF2. (D) qPCR detected the levels of USF1 in 157 OS samples. (E) qPCR examined USF1 levels in MG63 and 143B cells after various treatment. (F) Real-time assessed GAS6-AS2 levels in MG63 and 143B cells after various treatment. (G) Left: schematic representation of constructs for GAS6-AS2 promoter luciferase reporter. B1-B4: “JASPAR” predicted binding sequences of USF1 in GAS6-AS2 promoter. Luc: luciferase. Right: luciferase activities of 6 truncated constructs (site1-6) in HEK-293 T cells. (H) Luciferase activity detection. B2 WT: luciferase reporter containing wild-type “JASPAR” predicted binding sequence 2 of USF1. B2 MUT: luciferase reporter containing mutant-type “JASPAR” predicted binding sequence 2 of USF1. (I) ChIP assays. * P < 0.05, **P < 0.01.