Figure 1.
Stress-induced niche remodeling in O5AΔ/Δ mice. (A) Experimental design for IP injection of 5-Fluorouracil (5-FU) in coculture assay of MSPCs and LT-HSC. The following genotypes were analyzed by flow cytometry and IF 8 days after treatment: Control (CTRL): O5A+/+, n = 5, 5Afl/fl, n = 7 and O5AΔ/Δ, n = 10. (B) Protein content and polarity of LT-HSCs stained for CDC42-GTP (n = 30), measured by ImageJ software. (C) Number of colonies from 60 LT-HSCs after coculture for 6 days on MSPCs isolated from 5-FU injected mice with the following genotype: (CTRL): O5A+/+, n = 5, 5Afl/fl, n = 5 and O5AΔ/Δ, n = 10. (D) Primary transplantation (Tx) of 300 sorted LT-HSCs 8 days after 5-FU injection into lethally irradiated 129*Ly5.1 WT recipients. Experimental groups: CTRL: O5A+/+, n = 5 and 5Afl/fl, n = 4 and O5AΔ/Δ, n = 7. Graph showing the donor engraftment in PB. (E) Absolute numbers of donor-type LT-HSCs in the BM, 16 weeks after Tx. For flow cytometry gating strategy, see supplemental Figure 1. (F) Experimental design for IP injection of 5-FU in 8- to 10-week-old mice of following genotypes: CTRL: O5A+/+ n = 2, 5Afl/fl n = 2 and O5AΔ/Δ, n = 2. BM analysis 30 days after treatment. (G) Stacked whole-mount images from epiphyseal/metaphyseal BM. FABP4+ vasculature is shown in blue. CD150 is shown in red, other hematopoietic markers (CD41, CD48, and lineage) are shown in gray. The dashed lines denote the endosteum. Scale bar, 100 µm; n = 10 images (n = 6 images 5Afl/fl PBS) from 2 mice each group. The results represent 2 independent experiments. The graph showing the % of trabecular volume per BM volume. (H) Stacked whole-mount images from epiphyseal/metaphyseal BM. FABP4+ adipocytes are shown in red. Adipocytes are additionally marked by a yellow circle. Extended focus projection images. Scale bar, 100 µm; n = 10 images from 2 mice each group, femora. The graph on the right shows the number of adipocytes per image. (I) Experimental design for analysis of bone-digested stromal cells, isolated from compact bones, 8 days post 5-FU treatment. (J) Relative numbers of CD31+ ECs, OBCs, and immature MSPCs; flow cytometry gating strategy in supplemental Figure 1. *P < .05 (nonparametric Mann-Whitney test: B-G,J). The results represent 2 to 3 independent experiments. Data are represented as dots per mouse or cell and the mean ± SEM. Symbol legends as shown in Figure 1A.

Stress-induced niche remodeling in O5AΔ/Δ mice. (A) Experimental design for IP injection of 5-Fluorouracil (5-FU) in coculture assay of MSPCs and LT-HSC. The following genotypes were analyzed by flow cytometry and IF 8 days after treatment: Control (CTRL): O5A+/+, n = 5, 5Afl/fl, n = 7 and O5AΔ/Δ, n = 10. (B) Protein content and polarity of LT-HSCs stained for CDC42-GTP (n = 30), measured by ImageJ software. (C) Number of colonies from 60 LT-HSCs after coculture for 6 days on MSPCs isolated from 5-FU injected mice with the following genotype: (CTRL): O5A+/+, n = 5, 5Afl/fl, n = 5 and O5AΔ/Δ, n = 10. (D) Primary transplantation (Tx) of 300 sorted LT-HSCs 8 days after 5-FU injection into lethally irradiated 129*Ly5.1 WT recipients. Experimental groups: CTRL: O5A+/+, n = 5 and 5Afl/fl, n = 4 and O5AΔ/Δ, n = 7. Graph showing the donor engraftment in PB. (E) Absolute numbers of donor-type LT-HSCs in the BM, 16 weeks after Tx. For flow cytometry gating strategy, see supplemental Figure 1. (F) Experimental design for IP injection of 5-FU in 8- to 10-week-old mice of following genotypes: CTRL: O5A+/+ n = 2, 5Afl/fl n = 2 and O5AΔ/Δ, n = 2. BM analysis 30 days after treatment. (G) Stacked whole-mount images from epiphyseal/metaphyseal BM. FABP4+ vasculature is shown in blue. CD150 is shown in red, other hematopoietic markers (CD41, CD48, and lineage) are shown in gray. The dashed lines denote the endosteum. Scale bar, 100 µm; n = 10 images (n = 6 images 5Afl/fl PBS) from 2 mice each group. The results represent 2 independent experiments. The graph showing the % of trabecular volume per BM volume. (H) Stacked whole-mount images from epiphyseal/metaphyseal BM. FABP4+ adipocytes are shown in red. Adipocytes are additionally marked by a yellow circle. Extended focus projection images. Scale bar, 100 µm; n = 10 images from 2 mice each group, femora. The graph on the right shows the number of adipocytes per image. (I) Experimental design for analysis of bone-digested stromal cells, isolated from compact bones, 8 days post 5-FU treatment. (J) Relative numbers of CD31+ ECs, OBCs, and immature MSPCs; flow cytometry gating strategy in supplemental Figure 1. *P < .05 (nonparametric Mann-Whitney test: B-G,J). The results represent 2 to 3 independent experiments. Data are represented as dots per mouse or cell and the mean ± SEM. Symbol legends as shown in Figure 1A.

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