Fig. 6

Gcsf signaling was the downstream target of Rnf111-Smad2/3. (A) Real-time quantitative polymerase chain reaction (qPCR) of gcsfa, gcsfb and gcsfr in siblings and mutants at 3 dpf. The data are presented as mean ± standard deviation (SD) with *P<0.05, **P<0.01. (B) Whole-mount in situ hybridization analysis of gcsfr. (C) Real-time qPCR of gcsfr in HSPC of rnf111 morphants. The data are presented as mean ± SD with ***P<0.01. (D) Luciferase assay of gcsfr promotor in HEK293T cells. The data are presented as mean ± SD with *P<0.05, **P<0.01, ***P<0.001, NS: no significant difference. (E) Chromatin immunoprecipitation qPCR assay of Smad2-GFP binding to gcsfr promotor at 2 dpf. The data are presented as mean ± SD with *P<0.05, NS: no significant difference. (F) Rescue assay of gcsfb and gcsfr RNA in Rnf111 mutants. Red arrows indicate cmyb-positive hematopoietic stem and progenitor cells in the caudal hematopoietic tissue. All experiments were independently replicated at least three times. WT: wild-type; MO rnf111: rnf111 morphants; HSPC: hematopoietic stem and progenitor cell; IDE2: definitive endoderm 2 inducer; RING MU: Rnf111 RING mutant with the RING domain mutated; SIM MU: Rnf111 SIM mutant with three SUMO-interacting motifs all mutated; GFP: green fluorescent protein; MU: Rnf111 mutants with deletion of 4 base pair nucleotides; hpf: hours post-fertilization; dpf: days post-fertilization.