Figure 3.

Induced A1 knockdown does not affect the myeloid compartment. (a) Representative dot plots of single-cell suspensions derived from bone marrow from mice of the indicated genotypes kept on doxycycline for 17 days. Cells were stained with antibodies recognizing Gr-1 or Mac-1 cell surface markers and gated on forward/side scatter profiles and viable (Annexin V^-) cells prior analysis. (b) Quantification of data obtained in bone marrow (wt and single transgenic controls n=9; DTrRen n=6 DTrA1 n=8), spleen (wt and single transgenic controls n=7, DTrRen n=6, DTrA1 n=8) or peripheral blood (wt and single transgenic controls n=7, DTrRen n=5, DTrA1 n=7). Bars represent means±S.E.M. (c) Bone marrow-derived GFP⁺⁺ granulocytes were sorted and cultured for 72 h±G-CSF (50 ng/ml) and doxycycline (1 μg/ml), re-added after 40 h. Viability was assessed by flow cytometry and Annexin V exclusion. Bars represent means±S.E.M. All experiments were performed in technical duplicates (wt and single transgenic controls n=7; DTrA1 n=4). (d) Quantitation of A1 knockdown efficacy at the protein level in sorted Gr-1⁺ GFP⁺⁺ bone marrow cells derived from mice of the indicated genotypes kept on doxycycline for 17 days. Cells were cultured±G-CSF and 1 μg/ml doxycycline for 8 h. (e) Bone marrow cells were plated in Methocult medium in the presence of G-CSF or M-CSF plus IL-3 and IL-6. Cultures were maintained in the presence of doxycycline (1 μg/ml), which was re-added on day 3 and day 6. Colonies were scored blinded after 7 days. Data represent means±S.E.M. (Simultaneous addition: wt and single transgenic controls n=4; DTrA1 n=4). Alternatively, cytokines were added with a 24- h delay. Data represent means±S.D. (wt controls n=2; DTrA1 n=2). All experiments were performed in duplicates. ANOVA followed by Bonferroni post-hoc test was performed to evaluate significant differences. *P≤0.05, **P≤0.01 compared with pooled controls kept on doxycycline)