Fig. 1.

Panel (a) FB-KO mice are poor HSPC mobilizers. For mobilization studies mice (six animals per group) were mobilized with G-CSF (Amgen, Thousand Oaks, CA, USA) for 3 days (short mobilization) at 100 μg/kg per day by subcutaneous injection (SC) with AMD3100 (Sigma-Aldrich, St. Louis, MO, USA) for 1 day at 5 mg/kg by intraperitoneal injection (IP). At 6 h after the last G-CSF injection, 1 h after AMD3100 injection, the mice were bled from the retro-orbital plexus for WBCs analysis, and PB was obtained from the vena cava (with a 25-gauge needle and 1-ml syringe containing 250 U heparin) for SKL (Sca-1⁺/c-kit⁺/Lin⁻) cells, CFU-GM clonogenic progenitors and ELISA analysis. Mononuclear cells (MNCs) were obtained by hypotonic lysis of RBCs in BD Pharm Lyse buffer (BD Biosciences) as described. Results from two independent experiments are pooled together. *p ≤ 0.05. Panels (b) and (c) Impaired activation of the Nlrp3 inflammasome in mobilized FB-KO mice. Panel (b) The effect of G-CSF on Nlrp3 inflammasome activation was evaluated at the mRNA level in PB. The expression of NLRP3, ASC (Pycard), CASP1, IL-1β, IL-18, HMGB1, S100a9, AIM2, GSDMD, NLRP1a, and NLRP1b mRNAs in PB after G-CSF and AMD3100 mobilization was measured by qRT-PCR. Sequences of primers and amplification parameters employed for these studies are shown in ref. [11]. Panel (c) The effect of G-CSF on Nlrp3 inflammasome activation was evaluated at the protein level by ELISA in PB plasma. The C5a, IL-1β, IL-18, and Hmgb-1 levels were measured using enzyme-linked immunosorbent assay (ELISA) kits (C5a, Cloud-Clone cat. no. SEA388Mu; IL-1β, Cloud-Clone cat. no. SEA563Mu; IL-18, Affymetrix eBioscience cat. no. BMS618/3; HMGB-1, Cloud-Clone cat. no. SEA399Mu), according to the manufacuterers’ instructions. Results (absorbance) are presented as the percentage of control. Results from two independent experiments are pooled together. *p ≤ 0.01