Figure 4

STZ treatment leads to upregulation of SMAR1, p53 and GAD65 expression. A. RIN cells were treated with STZ in a dose dependent manner as indicated for 24 hrs followed by RT-PCR analysis of SMAR1, p53 and GAD65 mRNA expression. B. Western blot analysis of SMAR1, ser46 p53, ser15 p53, total p53 and GAD65 expression upon 4 mM STZ treatment in a time dependent manner. C. Same cells treated with 4 mM STZ were used for FACS analysis at indicated time points. D. RIN cells were treated with 4 mM of STZ and cells were harvested after indicated time points. The samples were than processed for ChIP analysis of GAD65 promoter upon STZ treatment for binding of p53, SMAR1, p300 along with various chromatin activation and repression markers. E &F. ChIP analysis of upstream promoter region of GAD65 (E) and actin (F) which were used as negative controls did not show any binding by SMAR1 or p53. The ChIP markers used represent activation (p300 & Ac H3K9) as well as repression (Me3H4K20 & HDAC1) of transcription.