BM homing of K3^−/− LSK cells. (A) Short-term homing assay with labeled BM LSK cells; mean percentages ± SD. **, P = 0.0046 by unpaired t test. n = 5–6 per genotype; 10 independent experiments. (B) Quantification of extravasated cells per 10⁵ injected LSK cells in BM by intravital two-photon microscopy 18 h after transplantation, given as mean value ± SD. *, P = 0.0159 by Mann-Whitney test. n = 4–5 per genotype; nine independent experiments. (C) Representative images illustrating transmigration of LSK cells up to 6 h after injection in Col2.3-GFP recipients. White (CMTMR), LSK cells; green (GFP), osteoblasts; blue (second harmonic signal), bone; and red (Q-Tracker 695 nm), blood. Arrowheads indicate LSK cells transmigrating across endothelium (white dashed lines); left panels, before transmigration; right panels, after transmigration. (D) LSK cells visualized in BM microvessels for up to 6 h after injection. Mean ± SD. ***, P = 0.0006 by unpaired t test. n = 5 per genotype; 10 independent experiments. (E) Firm adherent cells shown in D grouped according to their residence time on the BM endothelium and shown as frequencies ± SEM of the absolute number of visualized cells. ***, P < 0.0001 for <1 min; **, P = 0.0037 for >1 min; ***, P = 0.0008 for >10 min, by Fisher’s test. (F) Representative images of adherent LSK cells at the indicated time after transfer. Red (CMTMR), LSK cells; blue (second harmonic signal), bone; and green (FITC dextran), blood. Arrows, arrowheads, and asterisk indicate different single LSK cells, and the white dashed lines outline the endothelium. (G) Surface expression of integrins on LSK cells isolated from BM (top) or FL cells (bottom) is presented as histograms of the mean fluorescence intensity for the indicated anti-integrin mAb on K3^+/+ (blue lines) or K3^−/− cells (red lines). Isotype controls are indicated as shaded histograms. n = 3–4 per group; three to four independent experiments. (H and I) Adhesion analysis of sorted K3^+/+, with or without preincubation with an anti–α4 integrin–blocking mAb, and K3^−/− BM (H) or E14.5 FL LSK (I) cells under shear in microflow chambers precoated with rmE-selectin alone (E), rmE-selectin and VCAM-1, or rmE-selectin, CXCL12, and VCAM-1. Error bars represent the mean percentage of adherent cells to total LSK cells ± SEM. ns, P > 0.05; *, P < 0.05; **, P < 0.01 by Kruskal-Wallis test and Dunn’s multiple comparison post-test. n ≥ 4 per group; 10 independent experiments. ns, not significant. Bars, 50 µm.