Fig. 5

GADD45α is a direct target of miR-362-5p. a The relative expression of GADD45α mRNA was analysed by qRT-PCR, under different contexts, GAPDH was used as an internal control. Left panel: 293 T cells untransfected or transfected with a mimic control (mimic-ctrl) or a miR-362-5p mimic (mimic) as indicated. Right panel: BV173 and K562 cells were either left untransfected or were transfected with either a miR-362-5p inhibitor (inhibitor) or a miR-362-5p inhibitor control (inhibitor-ctrl), as indicated. b The protein expression of GADD45α was measured by Western blotting under the same conditions. Top panel of (b) shows the typical images of western blotting, and the bottom panel of (b) shows the respective quantifications. c Expression of GADD45α protein was examined by Western blotting in normal CD34⁺ cells, 293 T cells and 4 leukemia cell lines. Protein expression was quantified and normalized to β-actin. d Expression of GADD45α protein was examined by Western blotting in 3 healthy controls and 6 CML patients. Protein expression was quantified and normalized to β-actin. e Left panel: Sequence of the miR-362-5p binding sites within the human GADD45α 3’-UTR and a schematic diagram of the reporter constructs showing the wild-type GADD45α 3’-UTR sequence (wt) and the mutated GADD45α 3’-UTR sequence (mut). Shaded areas represent conserved complementary nucleotides of the miR-362-5p seed sequence in various mammals (Has: human, ptr: chimpanzee, Mml: rhesus, Mmu :mouse, Rno: rat, and cfa: dog) Middle panel: Luciferase activity assay in, 293 T cells, with cotransfection of the wt-or-mut-reporter and a miR-362-5p mimic (mimic) or a miR-362-5p mimic control (mimic-ctrl) as indicated. Right panel: Luciferase activity assay, as above, with an miR-362-5p inhibitor (inhibitor) and an miR-362-5p inhibitor control (inhibitor-ctrl) in BV173 and K562 cells . **P < 0.01