SF3B1 mutations perturb mitochondrial function. For all panels, isogenic iPSCs are abbreviated: normal (ISO), t(4;12);SF3B1 (t;SF3B1 or SF3B1), t(4;12);SF3B1;EZH2 (t;SF3B1;EZH2 or EZH2), and SF3B1 G742D-ablated (-G742D). (A) Erythroid differentiation of CD34-5F cells derived from MDS-RA1 patient iPSCs. Percentage of early (CD71⁺CD235a⁺) and mature (CD71^loCD235a⁺) erythroblasts, day 11 of differentiation. (B) Prussian blue iron staining of primary BMMCs from patient MDS-RA1 and erythroblasts from normal isogenic or t;SF3B1;EZH2 MDS-iPSCs. Ring sideroblasts were evaluated based on the criteria of >5 iron granules encircling at least one-third of the circumference of the nucleus. Scale bars 15 μm. (C) Frequency of annexin V⁺ apoptotic CD235a⁺ erythroid cells derived from isogenic iPSCs. (D) Total and active mitochondrial content in CD235a⁺ erythroid cells derived from isogenic iPSCs. (E) Total mitochondrial content measured as mean fluorescence intensity (MFI) of MTG in CD235a⁺ erythroid cells derived from isogenic iPSCs. (F) Ratio of active (measured as MFI of TMRE) to total (measured as MFI of MTG) mitochondria in CD235a⁺ cells derived from isogenic iPSCs. (G) Ratio of active to total mitochondria in normal (ISO) and t;SF3B1;EZH2 CD235a⁺ cells transduced with a control luciferase (+LUC) or EZH2 overexpression lentivirus (+EZH2). (C-G) Mean ± standard deviation of 3 experiments, 1 to 2 iPSCs per genotype. **P < .01, *P < .05, t test vs ISO unless indicated.