Figure S3.

Functional analyses of fetal and adult AMs and monocytes. (A) Cytokine expression in AMs from 6–12-wk-old Hem1^fl/flLysMCre^+/+ and LysMCre^+/+ mice after 24-h LPS (10 µg/ml) stimulation as measured by real-time PCR. (B) Flow cytometric histograms showing the relative cell numbers of AMs (gated as in Fig. S2 B) that have phagocytosed 1-µm fluorescent beads in BALF from Hem1^fl/fl LysMCre⁺ and LysMCre⁺ mice after 1 h of o.p. LPS followed by 30 min of o.p. bead exposure (MOI, 20). Bar graphs show the percentages of bead-positive AMs (left). Histogram of relative cell number of PMs (ghost⁻CD11b⁺F4/80⁺) taking up 1-µm fluorescent beads in peritoneal fluid from Hem1^fl/fl LysMCre⁺ and LysMCre⁺ mice after 1 h of i.p. bead injection (4 × 10⁶ particles; MOI, 20). Bar graph shows the percentages of bead-positive PMs (right). Data shown are mean ± SEM from n = 3–5 mice (6–12 wk old)/genotype; representative of 2–3 independent exps. *, P < 0.05; **, P < 0.01; Student’s two-tailed t test. (C) Purified monocytes (Monos) from PND2 LysMCre⁺ and Hem1^fl/flLysMCre⁺ migrate similarly in Transwell plates in response to CCL2 after 4-h stimulation. (D) Hem1^fl/flLysMCre⁺ and LysMCre⁺ control mice were infected with 2 × 10⁶ live Spn via oropharyngeal delivery and monocyte recruitment into BALF and measured at 48 h afterinfection (left); body weights were measured daily for 48 h after Spn infection (right). (E) Hem-1–deficient adult monocytes home normally to BALF 48 h after transfer. LysMCre⁺ and Hem1^fl/flLysMCre⁺ adult monocytes were purified (Purify mono) using magnetic beads. WT monocytes were labeled with CFSE, and Hem1^fl/fl LysMCre⁺ cells were labeled with CTV. Cells were mixed 1:1, and 2 × 10⁶ numbers of cells were injected i.v. Mice were then anesthetized; 5 × 10⁶ CFU Spn were delivered i.n. 48 h after infection; mice were euthanized and BALF collected. The percentage of CFSE⁺ or CTV⁺ of CD11b^hiLy6c^hi cells in BALF are shown. Data shown are mean ± SEM from n = 4–7 mice/genotype representative of >2 independent exps. *, P < 0.05; **, P < 0.01; Student’s two-tailed t test. cKO, conditional KO; FSC, forward scatter; KC, keratinocyte chemoattractant; Rel., relative; SSC, side scatter.