Fig. 5

Glutaredoxin 2 acts on CSPG4 transcription and migration via SP-1. A) Scheme of the trapping experiment revealing presence of SP-1 in the eluted substrate after western blot (WB) and mass spectrometry (MS). B) Electrophoretic mobility shift assays (EMSAs) using cell lysates of HeLa cells transfected with either control siRNA or Grx2 siRNA and probes consisting of the promoter or an enhancer region of the CSPG4 gene. C) EMSAs comparing SP-1 probe binding in HeLa lysates treated with or without 100 mM N-ethyl-maleimide. D) EMSAs of GBM-18 cell lysates transfected with either control siRNA or Grx2 siRNA. Shown is the binding of a CSPG4 promoter probe containing SP-1 binding sites during the shift assay and staining of SP-1 as well as Grx2 after western transfer of the same lysate. E) Quantification of D) and qRTPCR was performed to quantify GLRX2c transcription in the cells. F) EMSAs using extracts of HeLa cells transfected with either control siRNA or Grx2siRNA and the probe described in D). The levels of SP-1 and Grx2 were determined after western transfer using the same lysates. G) Quantification of GLRX2c and CSPG4 transcripts, as well as binding between SP-1 and probe in HeLa cells with decreased Grx2 levels compared to control cells. H) SP1-binding to the probe was plotted against GLRX2c transcripts based on all shift assays performed with HeLa, GBM-18, and U343-MGA cell lysates, R² = 0.543.