Knockdown of Bim expression leads to attenuation of apoptosis induced by JAK2 inhibition. (A) Annexin V apoptosis assay. WT HEL cells and HEL cells stably transfected with vector constitutively expressing either scrambled (pSuper-bim75, 1 stably transfected clone analyzed: sc) or shRNA sequences targeting Bim (pSuper-bim73; 3 stably transfected clones analyzed: sh#1, sh#2, sh#4) were treated with either DMSO or 3μM of JAK inhibitor I for 24 hours. Data are mean ± SD of annexin V⁺ cells from 5 independent experiments. Error bars represent SD. *P < .05. **P < .01. ***P < .001. N indicates not significant. (Inset) Expression analysis of knockdown of all isoforms of Bim (Bim_EL, Bim_L, Bim_S) in shBim #1, shBim #2, and shBim #4 cells compared with scrambled shRNA (sc) transfected cells by Western blot. (B) Flow cytometric analysis for Bax activation. The HEL cells stably transfected with shRNA targeting Bim (clone: shBim #1) or scrambled shRNA (sc) were treated with or without 3μM JAK inhibitor I for 18 hours. Data are results from a representative experiment repeated 3 times with similar results. (C) Flow cytometric analysis of the inner mitochondrial membrane potential (ΔΨm) breakdown. The HEL cells stably transfected with vectors constitutively expressing either shRNA targeting Bim (clone: shBim #1) or scrambled shRNA (sc) were treated with or without 3μM JAK inhibitor I for 24 hours. Data are results from a representative experiment repeated 3 times with similar results. (D) Colony-forming assay. The parental HEL cells (HEL), the HEL cells stably transfected with shRNA targeting Bim (clone: shBim #1; shBim), and scrambled shRNA (sc) were pretreated with JAK inhibitor I and plated in human MethoCult H4230. Data are mean plus or minus SD of colony numbers expressed as percentage of DMSO-treated cultures. Error bars represent SD (n = 3). **P < .01. ***P < .001.