Apoptosis levels in β₁^Δ/Δ cells in BM and spleen after PHZ treatment and the ability of β₁^Δ/Δ cells to home and form CFU-S. (A) Expression of activated caspase-3, an apoptosis marker, in the spleens of control and β₁^Δ/Δ mice. A significant proportion of apoptotic cells among splenocytes of β₁^Δ/Δ mice was noted after PHZ treatment. In contrast to β₁^Δ/Δ mice, no difference in apoptosis in spleen between control and α₄^Δ/Δ mice after PHZ treatment at day 4 was detected (data not shown). (B) Cells from BM and spleen of PHZ-treated (day 4) mice were put into in vitro culture made up of Iscove modified Dulbecco medium supplemented with 10% fetal bovine serum, l-glutamine, penicillin, and streptomycin containing cytokines (3 U/mL recombinant human Epo, 50 ng/mL recombinant murine stem cell factor, 10 ng/mL recombinant murine interleukin-6, and 10 ng/mL recombinant murine interleukin-3). Two days later, cells were washed, resuspended in the just described medium without the cytokines, and 6 hours later were taken for analysis. Caspase-3 expression in TER119^hi/CD71^hi cells is shown before (day 0) and after (day 2) in vitro incubation with cytokines and after cytokine withdrawal (6 hours). Note that in β₁^Δ/Δ mice, even at the initial time point, there is a difference in the level of apoptosis between BM and spleen, and this is exaggerated on cytokine deprivation. (C) Homing into nonirradiated recipients: Wild-type mice were injected with control (β₁⁺, n = 5 recipient mice) or β₁-deficient (β₁^Δ/Δ, n = 5 recipient mice) BM cells. Twenty-four hours later, recipients were killed and spleen cells were plated in methylcellulose cultures. Individually plucked colonies were genotyped (supplemental Figure 3) to determine the origin of the host (α₄^+/+, β₁^+/+) or donor (β₁^f/f, α₄^f/f, or β₁^Δ/Δ) colonies and proportion of homed progenitors. The number of assessed colonies is shown above the bars. (D and bottom panels) CFU-S assessment: Lethally irradiated wild-type recipients were injected with 250 000 control (β₁^f/f, α₄^f/f) or integrin-deficient (β₁^Δ/Δ, α₄^Δ/Δ) BM cells (5 recipients per group). Eleven days after transplantation, recipients were killed, spleens were harvested, fixed in Bouin solution, and macroscopic colonies under the spleen capsule were counted using a Nikon MZ6 dissecting microscope. The number of large colonies per spleen is shown. Appearance of CFU-S in recipients of control and integrin-deficient cells is also shown (lower panels). β₁^Δ/Δ mice have fewer CFU-S than controls, whereas α₄^Δ/Δ mice have more but usually of smaller size. Spleens were photographed in a 35-mm Petri dish with a Nikon Coolpix 995 camera. Images were uploaded to a computer and adjusted for brightness and contrast with Adobe Photoshop.