Fig. 4
IL-8 is the mediator of density related immune fluctuations. A. mRNA expression of soluble factors in Huh-7 cells with different cell densities; determination after 24 h of culture. B. IL-8 mRNA expression of Huh-7 cells at different cell densities, and IL-8 concentration of Huh-7 cells in culture medium of different cell densities; determination after 24 h of culture. C. Quantification of IL-8 protein expression in the 3D culture of Huh-7 cells with different cell densities; Image J was used for quantification; Three visual fields were randomly selected for counting in each sample; scale bar, 50 µm. D. Spearman correlation scatter plot of IL-8 expression and lymphocytes including Tregs and CD8 + T cells in HCC (n = 65). E. Gating strategy of flow cytometry analysis. F. Effect of coculture with gradient density Huh7 with IL-8 fluctuation on Tregs polarization in PBMCs; determination after 24 h of culture. G. Effect of coculture with IL-8 KD and control Huh-7 cells (1 × 105 cells/cm2) on Tregs polarization in PBMCs; IL-8 concentration in the culture medium of IL-8 KD and control Huh-7 cells (1 × 105 cells/cm2); determination after 24 h of culture. H. Effect of coculture with IL-8 OE and control HCCLM3 cells (2 × 105 cells/cm2) on Tregs polarization in PBMCs; IL-8 concentration in the culture medium of IL-8 OE and control HCCLM3 cells (2 × 105 cells/cm2); determination after 24 h of culture. I. Effect of recombinant IL-8 (10 ng/mL, 24 h) on Tregs polarization in PBMCs; determination after 24 h of culture. The data represent the mean ± S.D. of three independent experiments. Mann–Whitney U test or t-tests were used to compare the means of two groups. ANOVA were used for comparison among three groups. *P < 0.05; **P < 0.01; ***P < 0.001. Abbreviations: KD, knockdown; OE, overexpression; PBMCs, peripheral blood mononuclear cells