Quantification of cell death by flow cytometry. YO-PRO-1 and Hoechst33342 dyes were used for quantification of cell death. Y-axis indicates the percent of cell death indicated by YO-PRO-1 and Hoechst33342 double positive cells. The mean of duplicates is presented with error bars indicating variation from mean. (A) Levels of cell death in K562 cells when treated with EVs and EV RNA. Y-axis indicates percent cell death observed. The following treatments are presented: (Blue) untreated: K562 cells without any treatment; (green) K562 EV treated: K562 cells incubated with K562 EVs; (red) mock: K562 cells with Lipofectamine treated only (no RNA); (purple) K562 EV RNA treated: K562 cells treated with K562 EV RNA; (turquoise) complete scram 31-mer: k562 cells treated with 31-nt scrambled sequence. (B) Levels of cell death in BJ cells when treated with EVs and EV RNA. Y-axis indicates percent cell death observed. The following treatments are presented: (Blue) untreated: BJ cells without any treatment; (red) mock: BJ cells with lipofectamine treated only (no RNA); (green) BJ EV RNA: BJ cells transfected with BJ EV RNA; (purple) K562 EV RNA: BJ cells treated with K562 EV RNA; (light green) BJ EV: BJ cells incubated with BJ EVs; (turquoise) HeLa EV: BJ cells incubated with HeLa EVs; (orange) U2-Os EV: BJ cells incubated with U2-Os EVs; (light blue) MCF7 EV: BJ cells incubated with MCF7 EVs; (mauve) K562 EV: BJ cells incubated with K562 EVs. (C) Generality of RNY5 31-mer-induced cell-death phenotype. Bars indicate the net increase in cell death normalized to levels of cell death from mock treatment in each cell type. Four cancer cell lines including (blue) K562 (chronic myelogenous leukemia); (purple) HeLa (cervical adenocarcinoma); (red) MCF7 (breast adenocarcinoma); (green) U2-Os (osteosarcoma), and four primary cells including (light mauve) BJ (normal skin fibroblasts); (turquoise) HUVEC (normal human umbilical vein endothelial cell); (light blue) IMR90 (normal human lung fibroblasts); and (orange) HFFF (normal human fetal foreskin fibroblasts) were transfected with RNY5 31-mer. One hundred picomoles of RNY5 was used for each transfection, except (orange) HFFF, where 200 pmol of RNY5 31-mer was used. (D) Dose response curve of RNY5 31-mer-induced cell-death phenotype in BJ cells. The bars represent the percent of cell death when BJ cells are treated with increasing dose (10, 50, 100, 200, 300, and 400 pmol) of RNY5 31-mer or nonspecific RNA. AllStars negative control RNA (Qiagen) was used as a nonspecific RNA control. The levels of cell death in Untreated or Mock treated (Lipofectamine only) BJ cells are also indicated. (E) Levels of cell death in BJ cells from 100 pmol of synthetic RNA oligonucleotides transfection. Y-axis indicates the percent cell death. The synthetic RNA oligonucleotides used for transfection are as follows: (Blue) untreated: BJ cells without any treatment; (yellow) mock: BJ cells with Lipofectamine treated only (no RNA); (green) nonspecific RNA: nonspecific RNA control (AllStars negative control siRNA); (purple) 8-nt motif deleted: RNY5 sequence with nucleotides 14–21 motif deleted; (turquoise) RNY5 31-mer complement: 31-nt RNY5 3′ side fragment; (light orange) 8-nt motif scrambled: RNY5 31-mer sequence with nucleotides 14–21 scrambled; (blue) RNY5 31-mer scram: 31-nt completely scrambled sequence; (orange) DS RNY5 31-mer, double-stranded RNY5 31-mer duplex; (green) full-length RNY5: RNY5 83-mer full-length sequence; (light green) RNY5 31-mer: 5′ RNY5 31-nt fragment; (light purple) RNY5 23-mer: 5′ side RNY5 23-nt fragment. (F) Levels of cell death observed in K562 cells from 100 pmol of synthetic RNA oligonucleotides transfection. Y-axis indicates percent cell death. The synthetic RNA oligonucleotides used for transfection are as follows: (Blue) untreated: K562 cells without any treatment; (yellow) mock: K562 cells with Lipofectamine treated only (no RNA); (green) nonspecific RNA: nonspecific RNA control (AllStars negative control siRNA); (purple) 8-nt motif deleted: RNY5 sequence with nucleotides 14–21 motif deleted; (orange) 8-nt motif scrambled: RNY5 31-mer sequence with nucleotides 14–21 scrambled; (blue) RNY5 31-mer scram: 31-nt completely scrambled sequence; (red) DS RNY5: double stranded; (green) full-length RNY5 83-mer; (light green) RNY5 31-mer: 5′ RNY5 31-nt fragment.
