STAT3 is expressed in primary AML cells and plays a role in OXPHOS. (A) Western blot showing total and phosphorylated STAT3 levels in mononuclear cells derived from 2 cord blood (CB) and 18 human primary AML samples. (B) qPCR showing mRNA expression levels of STAT3 in 3 primary AML samples treated with scramble siRNA (siSCR) control or siRNA against STAT3 (siSTAT3) after 48 hours of culture. (C) Western blot showing STAT3 protein levels 48 hours after transfection of siSTAT3 compared with siSCR. (D) Seahorse Cell Mito Stress Test showing OCR at the time of highest STAT3 knockdown (24-48 hours) of a representative primary AML sample. (E) Combined data showing changes in basal OCR, ATP production, and maximal respiratory capacity in 4 primary AML samples, including sample 1D, upon STAT3 inhibition by siRNA. (F) Viability assay of STAT3-deficient cells compared with scramble control (siSCR) after 60 hours in culture (48 hours of siRNA KD plus 12 hours of viability). (G) Structure of the salicylamide STAT3 inhibitor SF25 (STAT3i). (H) Cell-based ELISA performed in HeLa cells showing cellular STAT1α, STAT3, STAT5A, and STAT5B binding to an immobilized DNA consensus sequence in the presence or absence of STAT3i as well as niclosamide. (I) Viability assay in 8 primary AML samples treated with various doses of STAT3i for 24 hours compared with vehicle control. (J) Maximal respiratory capacity changes based on Seahorse Cell Mito Stress Test in 3 primary AML samples upon treatment with 5 μM of STAT3i for 4 hours compared with vehicle control. Statistical analyses were performed using the Student t-test. P values are represented as follows: *P ≤ .05; **P ≤ .01; ***P ≤ .001; ****P ≤ .0001.