Expression of IL-1–signaling molecules and hematopoietic regulators in IL-1β–stimulated AGMs. (A) RT-PCR analysis of E11 AGM tissue before and after 3-day explant culture in the presence of 0, 1, or 10 ng/mL IL-1β. Changes in gene expression of some of the tested genes (Il1r1, Il1rap, Il1r2, Il1a, Il1b, Il18r1, Il18rap, Il18) are found after explant culture or are induced by the presence of IL-1β. (B) RT-PCR analysis of E11 AGM tissue before and after 3-day explant culture in the presence of 0, 1, or 10 ng/mL IL-1β. Changes in gene expression of some of the tested genes (Cxcl12a and Cxcl12b, Cxcr4, Mmp9) are found after explant culture or are induced by the presence of IL-1β. (C) Flow cytometric analysis of cultured E11 aorta explants for expression of CXCR4. E11 aorta explants were cultured in the presence of 0, 1, or 10 ng/mL IL-1β for 3 days before analysis. The percentage of CXCR4⁺ cells is indicated in the gated upper section (n = 3); 3 × 10⁴ events were analyzed, and 1.3 to 1.5 × 10⁴ events are shown. (D) RT-PCR analysis of AGM tissue from Il1r1^+/+ and Il1r1^−/− E11 embryos. AGM tissues before and after 3-day explant culture were used for RNA preparation. Changes in gene expression of some of the tested genes (Il81r1, Il81rap, Il18, Tnfr1, Tnfr2, Tnfa, Cxcl12a, Cxcl12b, Cxcr4, Mmp9) are found in the absence of IL-1RI directly or after explant culture; n = 2-3 for each gene. (E) RT-PCR analysis of UG26-1B6 cells treated with 10 ng/mL IL-1β for various times (2-24 hours) and examined for gene expression of several hematopoietic cytokines (Il6, Kitl) and chemokines (Cxcl12a, Cxcl2b). Representative experiments are shown in panels A, B, C, and E (n = 2). Actin was used as a cDNA normalization control. −RT indicates no reverse transcriptase; +RT, + reverse transcriptase.