Fig 1. STAT3 represses Jmjd3 expression in glioblastoma stem cells.

A. RT-qPCR of GS6-22 cells treated with S3I-201 or STA-21 demonstrates that Jmjd3 mRNA is upregulated at both 4 hours and 24 hours after inhibitor treatment. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (**p<0.01). B. Knockdown of STAT3 using an shRNA-containing lentivirus leads to the upregulation of Jmjd3 mRNA in GS6-22 cells, two days after selection. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (**p<0.01). C. Chromatin immunoprecipitation using anti-STAT3 antibody, followed by PCR using primers to the Jmjd3 promoter demonstrates STAT3 binding in GS7-2 and GS6-22 cells. Enrichment of DNA in STAT3 immunoprecipitation (relative to input) was quantified by ImageJ. D. Treatment of GS6-22 cells with retinoic acid increases Jmjd3 mRNA levels. GS6-22 cells were treated for 1 and 4 hours with 1 μM RA. Values represent the fold change relative to DMSO treated cells for three experiments; bars SD (*p<0.05, **p<0.01). E. S3I-201 treatment decreases NCor binding at the Jmjd3 promoter. GS6-22 cells were treated with S3I-201 (50 μM) or DMSO for 4 hours, fixed, lysed, and sonicated. Sonicated lysates were incubated overnight with anti-NCor or IgG control and subjected to chromatin immunoprecipitation. The resulting DNA was subjected to PCR using primers to the Jmjd3 promoter. F. Densitometry of E was performed using ImageJ.