Fig. 6.

Rescue of IL-10 and IL-13 eliminated the effects of let-7d in vitro. a Flow cytometry analysis showed that the percentage of CD204 positive cells was restored in the presence of purified IL-10 or IL-13 (100 ng/ml) respectively, as compared with control (PBS phosphate buffer solution). b The cell proliferation assay showed that the proliferation of HUVEC cells in conditioned medium from coculture system with let-7d-overexpressing RCC cells in the presence of 100 ng/ml IL-10 or IL-13 was significantly increased compared with that in the presence of control. The data represented the mean ± SD of three independent experiments with triplicates of each sample. *p < 0.05. Statistical significance was obtained using two way ANOVA. c Represented micrographs and quantitative data of a Boyden chamber assay showed that the migration of HUVEC cells was significantly restored in the conditioned medium from M0 macrophages cocultured with let-7d-overexpressing RCC cells in the presence of 100 ng/ml IL-10 or IL-13. Original magnification: ×40. d Photograph showed that the tubes formed by HUVEC cells following the addition of IL-10 or IL-13 in the coculture system. Bar, 500 μm. Histogram displayed the quantitative data of the number tubes formed by HUVEC cells in conditioned medium added with IL-10, IL-13 or control respectively. (c-d) The data presented are the mean ± SD of three independent experiments with five random fields counted for each chamber/well. e Real-time RT-PCR results showed that the expression of CD163, CD206, TGF-β and CCL22 were significantly increased and the expression of CD80, CD86, TNF-α, HLA-DR, IL-6 and IL-1β were significantly decreased in M0 cells cocultured with let-7d-overexpressing 786O after addition of 100 ng/ml IL-10 or IL-13 in the coculture system. The mRNA expression was normalized to GAPDH mRNA. The data represented the mean ± SD of three independent RT–PCRs (RNAs were from the same set). f ELISA assay showed decreased protein levels of TNF-α and IL-6 as well as increased protein level of TGF-β in conditioned medium from M0 cells cocultured with let-7d-overexpressing 786O after addition of 100 ng/ml IL-10 or IL-13 in the coculture system. g, h The expression of PIGF was significantly decreased in conditioned medium from M0 macrophages cocultured with let-7d-overexpressing 786O cells in the presence of control, compared with those in the presence of 100 ng/ml IL-10 or IL-13, at mRNA level (g) and protein level (h) as detected by real time RT–PCR and ELISA assay respectively. The data represented the mean ± SD of three independent experiments with triplicates of each sample. One-way ANOVA followed by the Bonferroni–Holm procedure. *p < 0.05, **p < 0.01, ***p < 0.001