Fig. 1: cMoP are dedicated progenitors for multinucleated giant cells.
From: Monocyte progenitors give rise to multinucleated giant cells
a Phalloidin (green) and Hoechst (blue) staining of a cMoP-derived MGC after stimulation with BCG-RFP (red). Scale bar: 20 µm. Channels were adjusted individually with respect to contrast and brightness. b Scheme of monocyte/macrophage cell development in murine bone marrow (BM). c FACS gating for sorting of cMoP, MDP, Lin− c-kit+ cells and MC from bone marrow of Cx3cr1gfp/+ mice. d Representative Hemacolor stainings of cMoP from Cx3cr1gfp/+ mice, stimulated with LM (Lipomannan from M. smegmatis, 4 µg/ml) for 2 or 6 days compared to M-CSF (50 ng/ml) controls. Scale bar: 100 µm. Gamma-correction was adjusted to 0.8 in all images. e cMoP (dark blue), MDP (cyan, dashed), Lin− c-kit+ cells (gray, dashed) and MC (light blue) were stimulated with LM (4 µg/ml) for 2, 4, and 6 days. Quantification of MGC per cm2 based on Hemacolor staining (compare Fig. 1d). Depicted are mean ± SEM of n = 3 (MDP D2 and D4), n = 4 (MDP D6), n = 4 (cMoP, MC, Lin− c-kit+ D2 and D4) and n = 6 (cMoP, MC, Lin− c-kit+ D6) biologically independent samples. ****p < 0.0001 (cMoP – MDP), ####p < 0.0001 (refers to MC and Lin− c-kit+); (two-way ANOVA, Tukey’s multiple comparisons test (MCT)). f cMoP (dark blue), MDP (cyan), Lin− c-kit+ cells (gray) and MC (light blue) from Cx3cr1gfp/+ mice were stimulated with LM (4 µg/ml) or BCG (MOI 20, MOI 100) for 6 days and quantified for MGC formation by Hemacolor staining. Bars show mean ± SEM of n = 13–15 (control), n = 4–6 (LM), n = 5–7 (BCG MOI 20) and n = 4 (BCG MOI 100) biologically independent samples examined over 3–9 experiments. Detailed numbers of samples (n) for each condition are presented in Supplementary Table 3. ns = not significant, ***p = 0.0003, ****p < 0.0001 (two-way ANOVA, Dunnett’s MCT). g Ly6C+ and Ly6C− blood monocytes (black) and BM-derived cMoP (dark blue) and MC (light blue) were stimulated with LM (4 µg/ml) for 6 days. MGC formation was quantified based on Hemacolor staining. Graph shows mean ± SEM of n = 11 biologically independent samples for cMoP and MC, and n = 2 biologically independent samples for blood monocytes. ****p < 0.0001 (student’s unpaired t-test, two-tailed).
