G csfr

HSCs were incubated with fluorescently labeled rLZ-8 in a 6-well plate containing DMEM medium (10% fetal bovine serum) for 30 min. Then, the cells were counted and inoculated on to the dried and ultraviolet-treated coverslips coated with polyethyleneimine at a density of 0.5×10⁵/cm², followed by PBST (PBS with 0.1% Tween 20) washes. Subsequently, the cells were fixed with 4% paraformaldehyde for 10 min at room temperature and permeabilized with PBS containing 0.5% saponin for 10 min. Then, the non-specific antibody binding was blocked by incubation with PBST containing 1% BSA and 22.52 mg/ml glycine for 30 min. Next, the cells were incubated with primary antibodies G-CSFR, CSF1R, GM-CSFRα, and GM-CSFRβ (Abcam, Cambridge, UK), respectively, in a humid chamber at 4°C overnight, followed by fluorescent-labeled secondary antibody in 1% BSA for 1 h in the dark. Finally, after washing with PBS, the cells on the coverslip were sealed on the glass slide to avoid drying and preserved at -20°C in the dark. The samples were observed under the Structured Illumination Microscope (SIM, DeltaVision OMX, GE Healthcare, Uppsala, Sweden).

Immunofluorescence staining was performed in accordance with standard protocols. Briefly, cells on slides were air-dried, then fixed in 4% paraformaldehyde overnight. Subsequently, endogenous peroxidases were inactivated by incubation in 3% H₂O₂–methanol at room temperature for 10 min. Slides were then blocked with goat serum sealant (KGSP03, Keygen, Jiangsu, China) at room temperature for 20 min. Immunofluorescence staining was performed using a primary antibody to G-CSF receptor (G-CSFR; 1:100 dilution, ab126167, Abcam, Cambridge, MA, USA) by incubation in a humidified environment for 2 h at 37℃. Secondary antibody detection was performed with goat anti-rabbit fluorescein isothiocyanate (1:100 dilution, 111-095-003, Jackson ImmunoResearch, West Grove, PA, USA) by incubation in darkness at 37℃ for 1 h. Slides were sealed by 5 min of incubation at room temperature with Vectashield mounting medium plus 4’,6-diamidino-2-phenylindole (DAPI) (KGA215, Keygen). Imaging was performed with a confocal microscope (BX43, Olympus, Tokyo, Japan).

JXT was purchased from Beijing Lvye Pharmaceutical Co. Ltd. (Beijing, China), Fujian (China) origin. JXT was cut into small pieces and soaked in 75% ethanol at 8 times volume overnight and then extracted 3 times with 1 h for each time. Finally, the filtrates were mixed and concentrated by a rotary evaporator with bath temperature lower than 40°C. Ethanol extract was lyophilized to powder.
The reagents and kits for determining enzyme activity were purchased from Nanjing Jiancheng Bioengineering Institute (NJBI, Nanjing, China). The antibodies for evaluating G-CSFR, Bcl-2, JAK2, pJAK2, STAT5a, pSTAT5a, STAT5b and pSTAT5b were purchased from Abcam (Abcam, USA). Amifostine was purchased from Dalian Merro Pharmaceutical Factory (Dalian, China); RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from HyClone (HyClone, USA). G-CSF ELISA kit was purchased from Shanghai Tongwei Biotech Co. Ltd. (Shanghai, China).