Figure 4.

Slit2 is preferentially expressed in GD-OF compared to fibrocytes. It is a TSH-inducible protein as a consequence of increased gene transcription and mRNA stability. (A). Three strains of confluent fibrocytes and GD-OF remained untreated or were treated with bTSH (5mIU/ml) for 6 h, RNA extracted, reversed transcribed, and cDNA subjected to real-time PCR for Slit2. (B) Fibrocytes and GD-OF were treated with bTSH (5mIU/ml) for the graded intervals indicated along the abscissa. Medium was collected and subjected to a specific Slit2 ELISA as described in Methods. (C) A 2970 bp fragment of the human Slit2 gene promoter was cloned as described in Methods and transfected into fibrocytes and GD-OF. Cell layers were assayed for luciferase activity. (D) Confluent GD-OF were untreated or treated with bTSH for 2 h and subjected to a ChiP transcription assay as detailed in Methods. (E) GD-OF were pre-treated with bTSH for 2 h. All cultures were treated at time “0” with DRB (20 µg/mL) without or in the continued presence of bTSH for the intervals indicated along the abscissa. RNA was extracted, reverse transcribed, and subjected to real-time quantitative PCR for Slit2. Values were normalized to their respective GAPDH levels. Slit2 ELISA, luciferase assay, and ChiP results were normalized to the respective protein levels. Data were expressed as the mean ± SD of triplicate determinations. *** p<0.001,** p<0.01. Experiments were performed three times.