Figure 3

GC-MSCs CM activates the STAT3 and ERK1/2 pathways in neutrophils. (a) Human promyelocytic leukemia HL-60 cells were induced to differentiate towards neutrophils by DMSO for 5 days. The percentage of CD11b-positive population was determined by using flow cytometry. (b) Neutrophilic-differentiated HL-60 cells were incubated with conditioned medium from GC-MSCs for different times. The levels of p-ERK1/2, p-STAT3, ERK1/2 and STAT3 were examined by using western blot. (c) Western blot analyses of ERK1/2 and STAT3 (T705) phosphorylation in neutrophilic-differentiated HL-60 cells that were stimulated with different GC-MSCs CM for 30 min. (d) Neutrophils were stimulated with GC-MSCs CM in the presence or absence of the ERK1/2-specific inhibitor U0126 (10 μM) or STAT3 inhibitor WP1066 (10 μM) for 30 min. The levels of p-ERK1/2 and ERK1/2 were examined by using western blot. (e) Neutrophils were harvested and stained with Annexin V/PI to evaluate the rate of apoptotic cells by using flow cytometry. *P<0.05. (f) Neutrophils stimulated with GC-MSCs CM in the presence or absence of the ERK1/2-specific inhibitor U0126 (10 μM) (g) or STAT3 inhibitor WP1066 (10 μM) (h) for 18 h. Total RNA was extracted from neutrophils and real-time PCR was performed using human-specific primers for the expression of IL-8, TNFα, and CCL2 mRNA