miR-24 mimic miR-24 inhibitor Control mimic   % positive SD P- value % positive SD P- value % positive SD P- value Unstimulated CD206 15.1 3.9 NS 18.8 2.7 NS 14.3 4.6 NS CD163 45.5 9.5 NS 39.5 9.9 NS 37.6 7.7 NS CD45 96.0 2.3 NS 95.1 2.8 NS 94.9 3.3 NS IFN-γ and TNF-α CD206 12.0 2.1 NS 8.7 1.7 NS 4.11 2.9 NS CD163 7.1 3.5 NS 6.3 4.1 NS 6.6 3.2 NS CD45 97.2 1.7 NS 96.5 1.6 NS 92.8 4.7 NS IL-4 and IL-13 CD206 51.2 9.2 * 30.9 6.8 NS 32.7 7.3 NS CD163 50.4 8.5 NS 52.7 6.2 NS 54.2 7.9 NS CD45 96.0 1.4 NS 96.2 1.5 NS 96.3 2.8 NS IFN-γ and TNF-α>IL-4 and IL-13 CD206 14.9 2.3 NS 10.0 1.5 NS 12.6 1.7 NS CD163 26.8 6.9 NS 33.7 6.1 NS 24.2 8.2 NS CD45 95.0 1.9 NS 97.5 1.4 NS 92.9 3 NS IL-4 and IL-13>IFN-γ and TNF-α CD206 36.8 6.5 * 9.4 2.5 NS 12.8 2.8 NS CD163 29.7 5.4 NS 25.6 3.4 NS 25.0 8.7 NS CD45 96.6 1.5 NS 90.5 2.0 NS 94.0 1.8 NS

Table 1: Overexpression of miR-24 in macrophages enhances CD206 expression during alternative activation and abrogates loss of CD206 expression during the transition from alternative to classical activation. Macrophages were transfected with miR-24 mimic, inhibitor, control mimic (all at 50 nM), or positive control (untransfected) for 18 h, and stimulated with either IFN-γ (10 ng/ml) plus TNF-α (1 ng/ml) or IL-4 (10 ng/ml) plus IL-13 (10 ng/ml) for 48 h; or IFN-γ (10 ng/ml) plus TNF-α (1 ng/ml)/IL-4 (10 ng/ml) plus IL-13 (10 ng/ml) for 24 h followed by replacement of the media with the opposing cytokine combination for a further 48 h, and expression of CD206, CD163 and CD45 was analysed by flow cytometry. Mean values, error bars (standard deviation (SD)), and significance compared to untransfected controls was calculated using Student’s t-test (two-tailed). n=4 biological donors. (*P<0.05). NS=not significant.