Inhibition of transcription blocks cytokine- but not ATP-mediated Microvesicle (MV) release. (A) CHME-5 cells were cultured in the presence or absence of actinomycin D (actD) (5 ng ml−1) starting 1 h before and during interferon (IFN)-γ or interleukin-4 (IL-4) treatments (20 ng ml−1, 24 h); ATP (1 mM, 30 min) was given to cells pretreated for 24 h with actD (ATP + actD on the graph) or not (ATP on the graph). MVs were then collected, stained with IB4-FITC, and analyzed by flow cytometry. Data are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). actD significantly reduced the release of MVs induced by cytokines but not that induced by ATP. Unpaired two-tailed Student’s t-test was used for statistical analyses (***p: 0.0009/p: 0.0002, respectively). (B) The release of MVs from cells treated with actD only was evaluated as in panel (A). Data are represented as fold increase compared to the control (not treated, NT on the graph) (mean ± SEM of four independent experiments). actD significantly reduced the “basal release” of MVs. Unpaired two-tailed Student’s t-test was used for statistical analyses (**p: 0.0086). (C) The heatmap ranks the transcriptional factors based on the strength of association with the transcripts co-regulated by IFN-γ and IL-4 treatments. Colored dots indicate the transcriptional factors that can be pharmacologically modulated. (D) CHME-5 cells were treated with cytokines (20 ng ml−1) for 24 h in the presence or absence of the AP-1 antagonist SR11302 (1 µM), the NFAT blocking peptide 11 R-VIVIT (1 µM) and the SP-1 inhibitor WP631 (100 nM); MVs were then analyzed by flow cytometry. Data are represented as fold increase compared to the untreated controls (mean ± SEM of four independent experiments). The color code used in the graph for the inhibitors is the same applied in panel (C) for labeling the corresponding targets. One-way ANOVA plus Dunnet’s post hoc test was used for analyzing the effects of SR11302 and 11R-VIVIT on cytokine-mediated MV release, while unpaired two-tailed Student’s t-test was used for WP631.