STAT3 mutations enhance proliferation and self-renewal. (A) Schematic overview of most common somatic STAT3 mutations.^12,23 (B) Immunoblotting of pY⁷⁰⁵STAT3, STAT3, V5, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from HPC7 cells expressing empty vector, STAT3, or STAT3 mutants (Y640F, S614R, and D661V). (C) Growth curves of HPC7 cells expressing either empty vector, STAT3, or STAT3 mutants (Y640F, S614R, and D661V) (mean ± standard deviation [SD], n ≥ 3). (D) Representative image of colony formation assay (CFA) of HPC^LSK expressing either empty vector, STAT3, or STAT3 mutants (Y640F, S614R, and D661V) (mean ± SD, n ≥ 3. (E) Representative images of colonies 10 days after plating derived from HPC^LSK CFA assay, at original magnification ×4. (F) Serial replating CFA from HPC7 expressing either empty vector, STAT3, or STAT3 mutants (Y640F, S614R, and D661V) (mean ± SD, n ≥ 3). (G) Immunoblot of HPC7 cells expressing either empty vector, STAT3, or STAT3^Y640 that were cytokine starved for 3 hours and then stimulated with IL-6 for 20 minutes (100 ng/mL), then washed with phosphate-buffered saline (PBS) and plated in cytokine-free media. Cells were harvested at the indicated time points and pY⁷⁰⁵STAT3, STAT3, V5, and GAPDH expression analyzed. Unpaired, 2-tailed Student t test was used in panel C, and 1-way analysis of variance was used in panel F for P value determination. ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ND, not determined; PTCL, peripheral T-cell lymphoma; stim, stimulation.