Fig. 2

miR-CVB3 + CpGMel significantly increases apoptosis, DAMPs release, and macrophage activation compared with miR-CVB3 and CpGMel. A Cell viabilities of 4T1 and MDA-MB231 cells after 24- and 48-h incubation with CpGMel, miR-CVB3, or miR-CVB3 + CpGMel (n = 5). B 4T1 cells were analyzed for annexin-V after 24-h incubation with different therapeutic regimens as indicated (n = 3–5). C 4T1 cells were treated as above, followed by immunostaining of calreticulin. The purple and blue fluorescences represent CRT and nucleus, respectively. Scale bars = 50 μm. D Quantified calreticulin by flow cytometry (n = 3–5). E HMGB1 level in the supernatant of 4T1 tumor cells after various treatments were measured by western blotting (left) and quantified using NIH ImageJ (right). F Measurement of extracellular ATP at 24 and 48 h after indicated treatments (n = 5). The level of extracellular ATP in the different treatment groups was normalized to that of the sham group and presented the differences as %. G Evaluation of macrophage maturation (CD80 and MHC-II-positive cells) using flow cytometry after incubation of macrophages with supernatant collected from 4T1 cells treated with PBS (sham), CpGMel, miR-CVB3, and miR-CVB3 + CpGMel, and non-treated (n = 3). The concentrations of each agent for cell treatment were as follows: miR-CVB3 at an MOI of 1, melittin at a concentration of 10 μg/ml, and CpG ODNs at a dose of 5 μg/ml. Data in this figure were analyzed by one-way ANOVA followed by Tukey’s test to determine differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001, ****, p < 0.0001)