Figure 4
The bioactivity of IFNγ secreted from IFNγ-ADSCs. A) The sensitivity of melanoma B16F10 cells to IFNγ was evaluated by the MTT cell viability assay. Melanoma cells were incubated in CM (culture medium) obtained from either EGFP-ADSCs (mock) or IFNγ-ADSCs for 3 days. Zeocin (104 μg/mL) and purified mouse IFNγ (1000 pg/mL) were used respectively as a positive and sample control (all samples are compared to mock). Cell viability after treatment is presented as a percentage relative to mock treated cells. ***P < .001. B) Representative FACS plots from Annexin V/propidium iodide (PI) apoptosis assay showing an increase in early apoptosis of melanoma cells when cells are incubated in CM from IFNγ-ADSCs (Lower panels) compared to CM from EGFP-ADSCs (Upper panels) for 12h and 24h incubation. One experimental representative of triplicates is shown. C) A bar graphs summarizing the FACS results from annexin V/PI apoptosis assay in two time points demonstrating statistically significant increase in early apoptosis of melanoma cells in the presence of IFNγ. Data represent mean values of three replicates in three independent experiments. ***P < .001. D) Monitoring the induction of the STAT1 and STAT2 components of Jak/STAT-mediated signal transduction pathways. Melanoma cells were stably modified to express Firefly luciferase (FL) under CMV enhancer-CAG promoter (FL-melanoma), or under direct repeats of the interferon-stimulated response element (ISRE) and a minimal TA promoter (ISRE/FL-melanoma). ISRE/FL-melanoma cells were incubated in CM obtained from either EGFP-ADSCs (mock) or IFNγ-ADSCs for 48 hours and then ONE-Glo™ luciferase assay reagent was added to each sample. Luminescence imaging showed IFNγ-induced Jak/STAT-mediated signal activation in melanoma B16F10 cells. (Left) FL-melanoma cells which were used as a positive control. (Middle) ISRE/FL-melanoma cells incubated with supernatants from EGFP-ADSCs. (Right) ISRE/FL-melanoma cells incubated with CM from IFNγ-ADSCs. One experimental representative of triplicates is shown.