Figure 3. Evidence for Increased Transcriptional Activity of the Y640F STAT3 Mutation.

A) Y640F mutant greatly increased STAT3 transcriptional activity compared to STAT3 wild type and other mutants. HEK293T cells were co-transfected with Cignal STAT3 reporter harboring an SIE response element upstream of luciferase reporter and vector alone (vector), wild type STAT3 (WT), or STAT3 mutants (S614R, Y640F, D661Y or D661V). (Bottom) Western blot to detect the expression of the different STAT3 variants using human STAT3 antibody and using antibody β-actin as loading control.

B) Quantitative RT-PCR indicating a reduction of the mir-223 precursor transcript in patient cells harboring the Y640F mutation. Relative expression is normalized to the mean of the Y640F sample group.

C) Western blot of MYC and E2F1 in leukemic LGL and normal unactivated control CD8+ (NCD8, negative control) and activated CD8+ cells (AcCD8, positive control). Two baseline patient samples from each mutation class are di splayed. Bar graphs depicts the average level of expression of E2F1 and MYC as a proportion of GAPDH, normalized to unactivated control CD8+ samples.

D) Response rates to Step 1 by mutation type as percent of total responding.