Figure 4. miR-328 Rescues Granulocytic Differentiation through Restoration of C/EBPα Expression.

(A) miR-328 levels in (left) 32Dcl3 cells undergoing G-CSF-induced differentiation; (middle) Lin⁻/Sca⁺/Kit⁺ HSC, CMP/GMP/MEP committed progenitors and mature neutrophil BM subpopulations from wild-type C57BL/6 mice (mean ± SEM); and (right) CD34⁺ human BM cells undifferentiated (white) and induced to differentiate for the indicated time toward the erythroid (light gray), megakaryocytic (dark gray), granulocytic (red), or monocytic (black) lineages (mean ± SEM).

(B) Wright-Giemsa-stained cytospins of G-CSF-treated (0–7 days) pSuper-, miR-328-, miR-328-Mut-, miR-223-, and/or miR-181b-infected 32Dcl3 and/or 32D-BCR/ABL cells (mean ± SEM). Levels of miR-223 in BCR/ABL⁺ cell lines and primary cells and effect of ectopic miR-223 on cell proliferation are reported in Figure S2.

(C) Wright-Giemsa-stained cytospins of primary G-CSF-treated (0–10 days) uninfected and miR-328-, miR-328-Mut-, and miR-223-infected CML-BC^CD34+ BM progenitors (mean ± SEM). For levels of ectopic miR-328 and miR-223 expression in primary CML-BC cells, see Figure S2.

(D) Myeloperoxidase (MPO) immunostaining of G-CSF-treated uninfected and miR-328- and miR-223-transduced CML-BC^CD34+ BM cells. Data are representative of three independent experiments (mean ± SEM).

(E) Western blot shows C/EBPα, hnRNP E2, and GRB2 levels in G-CSF-treated 32Dcl3 and empty vector-, miR-328-, miR-223-, miR-328-Mut-, and/or miR-181b-infected 32D-BCR/ABL and CML-BC^CD34+ BM cells (right).

(F) qRT-PCR shows levels of CEBPA in empty vector-, miR-223-, or miR-328-infected CML-BC cells (mean ± SEM). qRT-PCR showing CEBPA mRNA levels in empty vector-, miR-223-, or miR-328-transduced 32D-BCR/ABL cells (mean ± SEM) is reported in Figure S2.