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. 2017 Apr 4;8(20):33779–33795. doi: 10.18632/oncotarget.16810

Figure 2. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 and mitogen induced expression of immediate early response genes.

Figure 2

(A) SEL120-34A competitively inhibits CDK8 binding to ATP -desthiobiothin probes (ActivX) in KG-1 AML cell lysate; ERK1/2 was used as a control. CDK8 and ERK1/2 proteins in precipitates were revealed by WB. (B) SEL120-34A inhibits IFN-dependent phosphorylation of STAT1 S727. HCT-116 cells stimulated with IFNg for 4 h were treated with increasing doses of SEL120-34A, Flavopiridol (F) or vehicle DMSO (C) to determine changes in the phosphorylation of STAT1 S727 and Y701, as measured by WB. (*) unspecific band. (C) SEL120-34A inhibits serine phosphorylation of STAT1 S727 and STAT5 S726, whereas Ruxolitinib inhibits tyrosine phosphorylation of STAT1 Y701 and STAT5 Y694 in JAK2 V617F cells. SET-2 cells were treated with Ruxolitinib, SEL120-34A or vehicle DMSO (C) for 24 hours and protein levels of pS727, pY701 STAT1 and pS726, pY694 STAT5 were measured by WB. (D) SEL120-34A inhibits serum- induced mRNA expression of immediate early response genes in a dose- dependent manner.HCT-116 cells were synchronized in 0.5% FBS medium for 24 h, cells were pretreated for 120 minutes with 1 μM SEL120-34A followed by activation with 10% FBS and subsequent RNA extraction, DNAse I treatment and qRT-PCR reaction. The results were normalized for RPLP0 mRNA (means ±S.D., n=4). Statistical analysis of differences between mRNA levels for control and SEL120-34A at the indicated time point was performed using t-tests. A p value of ≤ 0.05 (*) was considered significant. (E) HCT-116 cells were prepared as in panel D and then challenged for 120 min with increasing doses of SEL120-34A, followed by the treatment with 10% FBS 45 min. RNA was extracted, DNAse I digested and subjected to the qRT-PCR reaction. The results were normalized for RPLP0 mRNA (means ±S.D., n=4).