miR-125b–mediated reduction of c-JUN and JUND protein expression may participate in miR-125b–induced block of granulocytic differentiation. (A) Predicted miR-125b binding sites within the CDS and UTR regions, and predicted shRNA target sites within the CDS regions of c-Jun (NM_010591.1) and Jund (NM_010592.4). (B) Western blotting of c-JUN (top panel) and JUND (bottom panel) in 32D/miR-ctrl and 32D/miR-125b cells. (C) Western blotting of c-JUN and JUND in 32D cells transduced with shRNA-ctrl, shRNA #1, and shRNA #2 lentiviruses. (D) Analysis of STAT3 DNA-binding in 32D/shRNA-ctrl, 32D/shRNA #1 and 32D/shRNA #2 cells after indicated time points of G-CSF stimulation. Fifteen μg of whole cell lysate was used. OD/G-CSF values normalized to OD/IL-3 are presented. (E) Proliferation kinetics of 32D cells expressing shRNA-ctrl (open circle), shRNA #1 (filled squares), and shRNA #2 (filled triangles) during G-CSF treatment. Mean of 4 (panels D-E) and a representative experiments of 4 are shown.