Exogenous miR-125b deregulates Bak1 protein. (A) An outline of luciferase reporter assay for validating the interaction of miR-125b with the 3' UTR of BAK1 is shown. Red text indicates the ''seed'' regions. In mutant reporter constructs, the MRE was deleted. (B) Repression of luciferase activity due to the interaction between miR-125b and the predicted MREs in the luciferase-Bak1-3' UTR constructs. The values represent the average ± SD (n = 3). *p < 0.05. (C) Western blot analysis of BAK1 expression in NB4 cells (left panel) or HL60 cells (right panel) after transfection with 100 nM miR-125b duplex or scrambled duplex. (D) The effects of suppression of miR-125b on Bak1 in mouse model. Upper: the overexpression of miR-125b was examined using qRT-PCR; lower: western blot analysis showed the Bak1 protein was repressed by miR-125b. lv-miR-125b: lentivirus vectors that expressed miR-125b; lv-NC: lentivirus vectors that expressed miR-NC, miRNA negative control. (E) Bak1 protein expression was inversely correlated with miR-125b levels in 33 of 47 pediatric APL patient samples. Red triangle: miR-125b expression (fold change vs. normal average); light blue diamond: Bak1 protein expression (fold change vs. normal average). Both miR-125b and Bak1 average expression levels of healthy donors were set at 1. (F) Western blotting images showing typical high and low Bak1 protein expression levels. The Bak1 protein level was quantified from western blot bands normalized to the β-actin level. The average expressions of Bak1 and miR-125b, presented as fold change compared with healthy samples, are listed in the table. (G) Bak1 protein expression in three normal donors using western blot (left), selected typical Bak1 protein expression in normal donor and pediatric patients with different ATRA responses was analyzed by western blot (right). N, normal donor; P, samples collected in primary diagnosis without treatment; CR, complete remission after therapy.