Fig. 3.

Exosomes shed from mutp53 tumor cells carry specific microRNA cargo. a Macrophages were co-cultured with HCT116 cells. RNA was extracted from the macrophages and subjected to qPCR analysis with primers specific to miR-1246. Values were normalized for RNU48 in the same sample. b Macrophages were grown in the presence of 10 µg exosomes isolated from HCT116 cells differing by their p53 status. miR-1246, miR-21, and miR-4454 levels were measured as in a. c M1 and M2 macrophages were transfected with LNA-miR-1246 mimic (mim) and compared with an equivalent control vector (con). RNA was extracted from the macrophages and subjected to qPCR analysis with primers specific to TNF-α and IL-10. d HCT116 mutp53 (R248W) cells were transfected with LNA-miR-1246 mimic (mim) or miR-1246 inhibitor (inhib.) and compared with an equivalent control vector (con). Forty-eight hours later, cells were co-cultured with M2 macrophages for an additional 3 days after which RNA was extracted from the macrophages and subjected to qPCR analysis with primers specific to TNF-α, CCL2, and IL-10. e Exosomes were isolated from HCT116 cells harboring mutp53 and separated from free proteins by a size-exclusion column. Macrophages were incubated with either pbs negative control (Con), the exosomes fraction (Exos) or the free proteins fraction (Prot.). RNA was extracted from the macrophages and subjected to qPCR analysis with primers specific to the indicated genes. Data are presented as fold changes compared with a PBS-treated control. f M2 macrophages were grown in the presence of 10 µg exosomes isolated from HCT116 cells differing by their p53 status for different time periods. RNA was extracted from the macrophages and subjected to qPCR analysis with primers specific to pre-miR-1246. All experiments in this figure were repeated three times, error bars represent standard errors