Fig. 5
Inhibition of Alox5 rescues the phenotype in Drosha cKO embryos. a A modest increase of Gata2 and Runx1 mRNAs relative to GAPDH in HSPCs (CD31+Kit+CD45−) from E11.5 cKO embryos compared to Ctr embryos were found by qRT-PCR analysis in triplicates (Mean ± SEM); p values were generated by unpaired Student t-test. Ctr: 12 embryos, cKO: 6 embryos. n = 3 litters. b qRT-PCR analysis of Alox5 and Alox5-AP mRNAs relative to GAPDH in ECs (CD31+Kit−CD45−, left) and HSPC clusters (CD31+Kit+CD45−, right) from E11.5 Ctr or cKO embryos is shown as Mean ± SEM; p values were generated by unpaired Student t-test. n = Ctr: 12 embryos, cKO: 6 embryos. 3 litters. c Schematic diagram of ex vivo zileuton rescue experiment (top panel). AGMs isolated from E9.5 (bottom left) or E10.5 (bottom right) Ctr or cKO embryos were incubated with 50 μM zileuton or vehicle for overnight, and all cells from AGMs were subjected to CFU assay. Total counts of colonies (BFU-E, CFU-GM, and CFU-GEMM) were presented as Mean ± SEM; p values were generated by unpaired Student’s t test . For zileuton treatment, n = Ctr: 11 embryos, cKO: 6 embryos. 3 litters for E9.5 and n = Ctr: 18 embryos, cKO: 9 embryos. 5 litters for E10.5. For vehicle, n = Ctr: 11 embryos, cKO: 3 embryos. 3 litters for E9.5 and n = Ctr: 18 embryos, cKO: 10 embryos. 6 litters for E10.5. d Schematic diagram of in vivo zileuton rescue experiment (top panel). Zileuton or vehicle 5 mg kg−1 was injected into pregnant mice at E9.5 and E10.5. At E11.5, AGM and FL were harvested and all cells from AGMs (bottom left) and FL (bottom right) were subjected to CFU assay in duplicates. Total counts of colonies (BFU-E, CFU-GM, and CFU-GEMM) were presented as Mean ± SEM; p values were generated by unpaired Student’s t test . For zilueton, n = Ctr: 11 embryos, cKO: 6 embryos. 4 litters. For vehicle, n = Ctr: 10 embryos, cKO: 5 embryos. 3 litters. e One percent of cells from E13.5 FLs were subjected to CFU assay. Total counts of colonies (BFU-E, CFU-GM, and CFU-GEMM) were presented as Mean ± SEM. Results were plotted as Mean ± SEM; p values were generated by unpaired Student’s t test . Drosha Ctr stands for Drosha Ctr (Drosha fl/fl or Drosha fl/+ or Drosha fl/+;Cdh5-Cre +) and Drosha cKO stands for Drosha cKO (Drosha fl/fl;Cdh5-cre +). Alox5 WT and Het stand for Alox5 +/+ and Alox5 +/−, respectively. Drosha Ctr;Alox5 +/+: n = 9 embryos. Drosha Ctr;Alox5 +/−: n = 8 embryos. Drosha cKO;Alox5 +/+: n = 3 embryos. Drosha cKO;Alox5 +/−: n = 4 embryos. 5 litters. NS, not significant
