Fig. 2

HCC-CAFs sustain neutrophil survival and activation.a, b Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 0, 1, 3, 6, or 12 h, the apoptosis of neutrophils was determined by using an Annexin V/PI apoptosis detection kit. Representative FACS plots of the apoptotic rate are shown in a. Frequencies of apoptotic neutrophils are shown in b. c, d Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 3 or 6 h. Total protein was extracted from neutrophils and the levels of Caspase 3 were detected by using western blotting. GAPDH was used as a loading control. c One representative experiment is shown. d The data indicate the mean ± SEM of three independent experiments, **P < 0.01. e Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 6 h, and total RNA was extracted from neutrophils and real-time PCR was performed to determine the mRNA levels of Mcl-1 and FAS, *P < 0.05. f, g Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 6 h. The expression of CD66b, PDL1, and CD62L on neutrophils was determined by flow cytometry. f One representative experiment is shown. g The data indicate the mean ± SEM of three independent experiments, **P < 0.01, *P < 0.05. h Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 6 h. Total RNA was extracted from neutrophils and real-time PCR was performed to determine them RNA levels of IL8, CCL2, and TNFa, ***P < 0.001, *P < 0.05. i Neutrophils were incubated in the presence or absence of HCC-CAFs CM for 48 h, and CM was collected and ELISA was performed to determine the protein levels of IL8, CCL2, and TNFa, ***P < 0.001, **P < 0.01