Fig 2: BC promotes the proliferation of mouse BMMNCs and the expansion of their HSPC population in vitro. (A,B) Flow cytometry analysis of FLT3 phosphorylation in mouse BMMNCs cultured in vitro with or without BC (10 μg/mL) treatment for 5–15 min. (A) Flow cytometry histogram showing the intensity of p−FLT3 Tyr591 versus cell counts. (B) The mean fluorescence intensity (MFI) of p−FLT3 Tyr591 from three independent experiments. (C) Cell counting of mouse BMMNCs cultured in vitro with or without BC treatment at different timepoints, which was presented as relative cell number to the cell number at day 0. Data represent three independent experiments. (D) Representative flow cytometry profiles to show the percentages of LSK (Lineage−Sca-1+cKIT+) in mouse BMMNCs cultured in vitro with or without BC treatment for 4 days or 7 days. Gates shown identify the LSK populations from Lineage− (Lineage negative cells) population. (E) The proportion and absolute number of LSK population in (D). Data represent three independent experiments. (F) Colony counting of HSPCs derived CFUs from 1 × 104 BMMNCs cultured in vitro with or without BC treatment for 4 days. CFU−GM: colony-forming unit of granulocyte and macrophage; BFU-E: burst−forming unit−erythroid; CFU−GEMM: colony−forming unit of granulocyte, erythrocyte, monocyte and megakaryocyte. Data represent triplicate experiments. All data are shown as mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t test).

Fig 3: BC increases the proportion of primitive HSC population and colony-forming ability of human CD34+ cord blood cells in vitro. (A,B) Flow cytometry analysis of FLT3 phosphorylation in CD34+ UCB cells cultured in vitro with or without BC (10 µg/mL) treatment for 2–15 min. (A) Flow cytometry histogram showing the intensity of p-FLT3 Tyr591 versus cell counts. (B) The mean fluorescence intensity (MFI) analysis of p-FLT3 Tyr591 from three independent experiments. (C) Representative flow cytometry profiles to show the percentage of CD34+CD38- and CD34+CD38-CD45RA-CD90+ cells in CD34+ UCB cells cultured in vitro with or without BC treatment for 4 days or 7 days. Gates shown identify the CD34+CD38- and CD34+CD38-CD45RA-CD90+ populations from CD34+ cells. (D,E) The proportion and absolute number of CD34+CD38- and CD34+CD38-CD45RA-CD90+ cells in (C). Data represent three independent experiments. (F) Colony counting of HSPCs derived CFUs from 2 × 103 CD34+ UCB cells cultured in vitro with or without BC treatment for 7 days. Data represent triplicate experiments. All data error bars are shown as mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t test).

Fig 4: BC administration increases the proportion of HSPC population and colony-forming ability of BMMNCs in healthy mice. (A) Experimental schematic of BC administration in healthy mice. C57BL/6 mice were administered once daily with BC (10 or 20 mg/kg) or vehicle by intraperitoneal (i.p.) injection for 7 days (n = 5). Mice were sacrificed 24 h after the last injection. B6 represents C57BL/6 mice. Red arrows indicate BC injection timepoints. (B) Histological analysis of spleen tissues by hematoxylin and eosin (HE) staining. Scale bar: 200 µm. Black arrows indicate the white pulp of spleen. (C) Representative flow cytometry profiles showing the percentage of LSK (left), LT-HSCs (LSKCD34-FLT3-), ST-HSCs (LSKCD34+FLT3-) and MPPs (LSKCD34+FLT3+) (right) in BMMNCs from control or BC (10 or 20 mg/kg) treated mice. Gates shown identify the LSK populations from Lineage- (Lineage negative cells) and the LT-HSC, ST-HSC and MPP populations from LSK cells. (D) The proportions and absolute number of LSK, LT-HSCs, ST-HSCs and MPPs in BMMNCs from control or BC (10 or 20 mg/kg) treated mice in flow cytometry analysis as shown in (C) (n = 5). (E) The numbers of CFUs formed by BMMNCs from control or BC (10 or 20 mg/kg) treated mice (n = 3). All data are shown as mean ± SEM, * p < 0.05, ** p < 0.01 (Student’s t test) versus control.

Fig 5: BC interacts with STS1 and STS2 HP domains and promotes phosphorylation of FLT3 and cKIT by inhibiting STS1 and STS2. (A,B) Molecular docking analysis of the binding of BC with STS1 (A) or STS2 (B). Left: The representative docking pose of BC with the HP domain STS1 (5VR6) or STS2 (5WDI). BC and the key residues of protein receptor are shown as sticks. The red dashed lines indicate the hydrogen bonds formed. Right: The 2D docked conformation showing the hydrophobic contacts (red eyelash shape) between BC and the HP domain of STS1 or STS2. (C,D) Capillary electrophoresis analysis of the binding of BC to STS1 (C) or STS2 (D). BC, STS1 or STS2 samples were prepared in 20 mM PBS at pH 7.0, and electrophoresis was run under 20 kV voltage. Red (C) or blue (D) arrows represent STS1 or STS2 protein peaks, respectively. (E,H) HEK293T cells were transiently transfected with FLT3 (E) or cKIT (H) constructs, and 48 h later, cells were stimulated with 10 µg/mL BC for different periods of time at 37 °C. Phosphorylated FLT3 (p-FLT3) or cKIT (p-cKIT) and endogenous STS1/STS2 proteins were detected by Western blot (n = 3). GAPDH was used as internal control. (F,G) FLT3 construct was transfected into HEK293T cells together with or without pcDNA-STS1 (F) or -STS2 (G). Then, 48 h later, cells were stimulated with BC for 2 min at 37 °C. The overexpressed proteins and phosphorylated FLT3 were detected by Western blot (n = 3). Vec indicates empty vector control. (I,J) cKIT construct was transfected into HEK293T cells together with or without Flag-tagged STS1 (I) or STS2 (J). Then, 48 h later, cells were stimulated with BC for 2 min at 37 °C. The overexpressed proteins and phosphorylated cKIT were detected by Western blot (n = 3). Vec indicates empty vector control. All data are shown as mean ± SEM, ** p < 0.01 (Student’s t test).