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. 2016 Nov 23;7(52):87016–87030. doi: 10.18632/oncotarget.13522

Figure 6. Effects of 4LB5-HP-RNase on cellular and EV-associated miRNAs.

Figure 6

(A) NCL-dependent mature miR-21, -221 and -222 levels were analyzed by Real-Time PCR after 72 h of incubation of MDA-MB-231 cells with increasing concentrations (6–100 nM) of 4LB5-HP-RNase. (B) Comparison between the effects of 4LB5 (grey bars) and 4LB5-HP-RNase (white bars) on mature miR-21, -221 and -222 levels by using an equimolar concentration (25 nM) of compounds for the treatment of MDA-MB-231 cells. Relative microRNA expression levels were normalized for control untreated sample, and relative abundance (fold change) is reported. Data are the average of three independent experiments performed in triplicate. Mean + SD is reported. (C) The mature miR-21 levels were analyzed by Real-Time PCR on RNA extracted from exosomes derived from the conditioned medium of SW620 cancer cells treated for 72 h with equimolar doses (50 nM) of 4LB5-HP-RNase (white bars) or 4LB5 (grey bars), tested in a parallel assay. Relative microRNA expression levels were normalized for control untreated sample, and relative abundance (fold change) is reported. (D) The snoRNA RNU48 was used as negative control of RNAs cellular contamination. (E) Western Blotting analysis showing NCL in both total lysates and MVs from SW620 cells, used for the experiments reported in C and D.