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. 2018 Jan 29;9:28. doi: 10.3389/fimmu.2018.00028

Figure 5.

Figure 5

Extracellular vesicles (EV) derived from Poly(I:C)-stimulated U937 cells have antiapoptotic effects in synovial fibroblasts. (A) Percentage of apoptotic Annexin V-positive synovial fibroblasts upon treatment with U937 cell-derived EV, as measured by flow cytometry (representative flow cytometry charts are provided as Figure S5C in Supplementary Material). Supernatants from the last washing step of Poly(I:C) EV pellets and fetal calf serum EV control (FCS EV) were used to control for Poly(I:C) carryover and the effects of potential residual FCS-derived EV, respectively. (B) The cleavage of caspase 3 in synovial fibroblasts treated for 24 h with TRAIL ± U937 cell-derived EV. Shown is Western blot for caspase 3 and cleaved caspase 3 from one of n = 7 biological replicates with α tubulin as loading control. The nitrocellulose membrane from the same gel was used for detection of caspase3, cleaved caspase 3 and α tubulin with different exposure times for detection of each protein. Provided is merged image and the original gel images are provided as Figure S9 in Supplementary Material. Densitometry analysis of the cleaved caspase 3 bands was normalized to uncleaved caspase 3 bands. (C) Percentage of apoptotic Annexin V-positive synovial fibroblasts upon treatment with TRAIL ± U937 cell-derived EV or supernatants from the last washing step of Poly(I:C) EV pellets (supernatants), as measured by flow cytometry (representative flow cytometry charts are provided as Figure S6A in Supplementary Material). Shown are two different experimental setups with n = 9 and n = 16 biological replicates. (D) Percentage of apoptotic Annexin V-positive synovial fibroblasts, as measured by flow cytometry, upon treatment with TRAIL ± peripheral blood mononuclear cell (PBMC)-derived EV or supernatants from the last washing step of Poly(I:C) EV pellets (supernatants). Representative flow cytometry charts are provided as Figure S7A in Supplementary Material. PBMCs from healthy donors were cultured ex vivo in the presence or absence of Poly(I:C) for 16 h. Heatmap (row scaling) shows the clustering of TRAIL-induced apoptotic responses of synovial fibroblasts (SFs) under different experimental conditions based on the flow cytometry measurements of Annexin V binding. (E,F) The activity of NF-κB in synovial fibroblasts cocultured with U937 cell-derived EV ± TRAIL for 6 h, as measured by a Dual Luciferase Reporter Assay System. Data are expressed as the ratio of the activity of Firefly to Renilla luciferase in cells transfected with pRL_GAPDH plus pGL4.32[luc2P/NF-κB-RE/Hygro] or pGL4.27[luc2P/minP/Hygro] vectors for 30 h. Shown are two different experimental setups: (E) a screening experiment with n = 2 biological replicates and (F) a confirmatory experiment with n = 6 biological replicates. (G) Percentage of apoptotic Annexin V-positive synovial fibroblasts, as measured by flow cytometry, upon treatment with TRAIL ± Poly(I:C) EV and/or sc-514 (50 µM), the inhibitor of IKK-2, shown are biological replicates. Representative flow cytometry charts are provided as Figure S8A in Supplementary Material. Statistics: (A) one-way ANOVA with Dunnett’s multiple comparisons test and Geisser–Greenberg correction for unequal variances (B,C,F) Friedmann ANOVA with Dunn’s multiple comparisons test (G) one-way ANOVA with Tukey’s multiple comparisons test and Geisser–Greenberg correction for unequal variances. ns, not significant.