Figure 2. The proteomic impact of deleting mir-223 in mouse neutrophils.

a, Schematic of neutrophil labelling and analysis. Haematopoietic progenitors were isolated from wild-type (WT) or mir-223^-/Y (KO) male mice and cultured in SILAC media containing granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) for six days. To enhance differentiation, SCF was withdrawn over the next 42 h. Mature neutrophils were mixed, and proteins were size-fractionated for quantitative MS analysis. mRNA was also collected from the cultures and directly from mice for expression profiling. b, mir-223 expression detected with RNA blots probing for miR-223. One blot analysed total RNA from sorted subpopulations of cells cultured in vitro (left, with sorting profiles shown at the far left). The other blot analysed total RNA from cells cultured in vitro for eight days and from neutrophils isolated directly from bone marrow (right). As a loading control, blots were re-probed for U6 small nuclear RNA. Below each blot are the relative expression levels, normalized using the loading control. Radio-labelled RNA markers (M) are also shown. c, Analysis of neutrophils isolated directly from mice (left) and those derived in vitro from haematopoietic precursors (right), monitoring the effects of miR-223 loss on messages with single miR-223 sites in their 3′ UTRs. Plotted is the fraction of messages that changed at least to the degree indicated on the x axis, otherwise as in Fig. 1c. d, The fraction of upregulated proteins deriving from messages with miR-223 7-8mer 3′-UTR sites, plotted as in Fig. 1b. e, The impact of deletion of mir-223 on neutrophil proteins, considering proteins from messages with single miR-223 sites in their 3′ UTRs, plotted as in Fig. 1c. f, Targeting efficacy in ORFs (n = 69) and 3′ UTRs (n = 50), plotted as in Fig. 1e. g, Efficacy of single 7-8mer sites and multiple sites, plotted as in f.