Figure 5.
Engagement of SDF1/CXCR4 is sufficient to induce HSPC polarization.(A) Localization of ligand-receptor pairs in polarized HSPCs. Selected Z stacks of confocal images of representative cells. White arrows highlight the point of contact with the stromal cell. Actin appears in red and DNA in blue. Left: VCAM-1 and VLA-4 appear in green. Inverted images of VCAM-1 and VLA-4 are presented. Middle: ICAM-1 and LFA-1 appear in green. Inverted images of ICAM-1 and LFA-1 are presented. Right: CXCR4 and SDF1 appear in green. Inverted images of CXCR4 and SDF1 are presented. Scale bars = 5 µm. (B) Schematic illustration of an HSPC in a microwell coated with protein A (light green) and ligand of interest (dark green). Potentially engaged receptor is depicted in red. (C) Representative inverted images of microtubules highlighting the shape of HSPCs cultured in uncoated (PAA), SDF1-, ICAM-1–, or VCAM-1–coated microwells. Scale bar = 5 µm. (D) SuperPlot of the aspect ratios of the HSPCs cultured in microwells coated with the molecules of interest. The cell aspect ratio was calculated as the ratio of the lengths of the short and long axes of the cell. Each color represents a biological replicate (three replicates: PAA ntotal = 107, SDF1 ntotal = 228, ICAM-1 ntotal = 182, and VCAM-1 ntotal = 221). For each replicate, the median appears as a large diamond, triangle, hexagon, or disk with the corresponding color. The mean of the medians appears as a black bar. ***, P = 0.0006; Kruskal–Wallis ANOVA. (E) SuperPlot of the polarization index of HSPCs cultured in microwells coated with the molecules of interest. Each color represents a biological replicate (three replicates: PAA ntotal = 107; SDF1 ntotal = 228; ICAM-1 ntotal = 182, and VCAM-1 ntotal = 221). For each replicate, the median appears as a large diamond, triangle, hexagon, or disk with the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (F) Orthogonal view of representative images of an HSPC cultured in microwells coated with PAA, SDF1-FC alone, and SDF1-FC in the presence of AMD3100 (100 µM). Actin appears in red, the centrosome in white, DNA in blue, and SDF1-FC in green. Scale bar = 5 µm. (G) SuperPlot of the polarization index of HSPCs cultured in microwells coated with PAA, SDF1-FC alone, and SDF1-FC in the presence of AMD3100 (100 µM). Each color represents a biological replicate (four replicates: PAA ntotal = 203; SDF1 ntotal = 231; and SDF1-FC + AMD3100 (100 µM) ntotal = 219). For each replicate, the median appears as a large diamond, triangle, or square with the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (H) Representative images of osteoblasts (hFOB), endothelial cells (HUVEC), and fibroblasts (BJ) immunostained for SDF1 (in green). Actin appears in red and DNA in blue. Scale bar = 20 µm. (I) Upper panel: Representative immune blot performed on osteoblasts, endothelial cells, and fibroblast lysates, using anti-SDF1 and anti-GADPH antibodies. Lower panel: Quantification of the immunoblots from three independent experiments. The ratio of signal intensities of SDF1 on GADPH are normalized to the value obtained in osteoblasts. Mean values are presented. Errors bars are SD. *, P = 0.036; **, P = 0.006; unpaired t test. (J) SuperPlot of the polarization index of HSPCs cultured on osteoblasts in the absence (CTL) or presence of AMD3100 (100 µM). Each color represents a biological replicate (four replicates: CTL ntotal = 249 and AMD3100 ntotal = 266). For each replicate, the median appears as a large diamond, or triangle with the corresponding color. The mean of the medians appears as a black bar. ***, P = 0.0003; Mann–Whitney U test.

Engagement of SDF1/CXCR4 is sufficient to induce HSPC polarization. (A) Localization of ligand-receptor pairs in polarized HSPCs. Selected Z stacks of confocal images of representative cells. White arrows highlight the point of contact with the stromal cell. Actin appears in red and DNA in blue. Left: VCAM-1 and VLA-4 appear in green. Inverted images of VCAM-1 and VLA-4 are presented. Middle: ICAM-1 and LFA-1 appear in green. Inverted images of ICAM-1 and LFA-1 are presented. Right: CXCR4 and SDF1 appear in green. Inverted images of CXCR4 and SDF1 are presented. Scale bars = 5 µm. (B) Schematic illustration of an HSPC in a microwell coated with protein A (light green) and ligand of interest (dark green). Potentially engaged receptor is depicted in red. (C) Representative inverted images of microtubules highlighting the shape of HSPCs cultured in uncoated (PAA), SDF1-, ICAM-1–, or VCAM-1–coated microwells. Scale bar = 5 µm. (D) SuperPlot of the aspect ratios of the HSPCs cultured in microwells coated with the molecules of interest. The cell aspect ratio was calculated as the ratio of the lengths of the short and long axes of the cell. Each color represents a biological replicate (three replicates: PAA ntotal = 107, SDF1 ntotal = 228, ICAM-1 ntotal = 182, and VCAM-1 ntotal = 221). For each replicate, the median appears as a large diamond, triangle, hexagon, or disk with the corresponding color. The mean of the medians appears as a black bar. ***, P = 0.0006; Kruskal–Wallis ANOVA. (E) SuperPlot of the polarization index of HSPCs cultured in microwells coated with the molecules of interest. Each color represents a biological replicate (three replicates: PAA ntotal = 107; SDF1 ntotal = 228; ICAM-1 ntotal = 182, and VCAM-1 ntotal = 221). For each replicate, the median appears as a large diamond, triangle, hexagon, or disk with the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (F) Orthogonal view of representative images of an HSPC cultured in microwells coated with PAA, SDF1-FC alone, and SDF1-FC in the presence of AMD3100 (100 µM). Actin appears in red, the centrosome in white, DNA in blue, and SDF1-FC in green. Scale bar = 5 µm. (G) SuperPlot of the polarization index of HSPCs cultured in microwells coated with PAA, SDF1-FC alone, and SDF1-FC in the presence of AMD3100 (100 µM). Each color represents a biological replicate (four replicates: PAA ntotal = 203; SDF1 ntotal = 231; and SDF1-FC + AMD3100 (100 µM) ntotal = 219). For each replicate, the median appears as a large diamond, triangle, or square with the corresponding color. The mean of the medians appears as a black bar. ****, P < 0.0001; Kruskal–Wallis ANOVA. (H) Representative images of osteoblasts (hFOB), endothelial cells (HUVEC), and fibroblasts (BJ) immunostained for SDF1 (in green). Actin appears in red and DNA in blue. Scale bar = 20 µm. (I) Upper panel: Representative immune blot performed on osteoblasts, endothelial cells, and fibroblast lysates, using anti-SDF1 and anti-GADPH antibodies. Lower panel: Quantification of the immunoblots from three independent experiments. The ratio of signal intensities of SDF1 on GADPH are normalized to the value obtained in osteoblasts. Mean values are presented. Errors bars are SD. *, P = 0.036; **, P = 0.006; unpaired t test. (J) SuperPlot of the polarization index of HSPCs cultured on osteoblasts in the absence (CTL) or presence of AMD3100 (100 µM). Each color represents a biological replicate (four replicates: CTL ntotal = 249 and AMD3100 ntotal = 266). For each replicate, the median appears as a large diamond, or triangle with the corresponding color. The mean of the medians appears as a black bar. ***, P = 0.0003; Mann–Whitney U test.

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