Fig. 2

TRAIL expression and modulation in pIL6-TRAIL ⁺ -GFP ⁺-UC-MSCs. a Left: transfection efficiency of UC-MSCs evaluated by flow cytometry for GFP fluorescence. Wildtype UC-MSCs were negative as compared to GFP ⁺-UC-MSCs (red histogram: 83.5%) in a similar amount as for pIL6-TRAIL ⁺ -GFP ⁺-UC-MSCs (blue histogram: 87.6%). Right: FACS analysis revealed constitutive TRAIL expression in transduced UC-MSCs (green histogram: 62.5%) that was significantly enhanced by the conditioned medium from U-266 cells (blue histogram: 80.3%) as well as in response to IL-1α/IL-1β (red histogram: 91.2%). b Confocal microscopy of pIL6-TRAIL ⁺ -GFP ⁺-UC-MSCs confirmed the upregulation of TRAIL (FITC, green) induced by either U-266 supernatant or IL-1α/IL-1β treatment. Phalloidin staining was used to show actin (TRITC-red); nuclei were counterstained with Hoechst 33342 (blue). Magnification: 100×. c Evaluation of TRAIL expression in transduced cells by RT-qPCR, western blot analysis, and ELISA. (i) After either U-266 or IL-1α/IL-1β treatment, TRAIL mRNA levels in pIL6-TRAIL ⁺ -GFP ⁺-UC-MSCs increased up to 2.2-fold and 2.5-fold compared to basal condition. Data are relative amount of mRNA expression normalized to GAPDH and are presented as mean ± SD of three independent experiments. *p < 0.05. (ii) Western blot analysis of lysates from transduced cells showing increased levels of TRAIL protein after U-266 medium or IL-1α/IL-1β conditioning. GAPDH was used as loading control. (iii) ELISA measurement of soluble TRAIL in supernatants of pIL6-TRAIL ⁺-GFP ⁺-UC-MSCs after 24 h of treatment with both U-266 conditioned medium and IL-1α/IL-1β. In both instances the increase was significant as compared to control cells (p < 0.05). GFP green fluorescent protein, MSC mesenchymal/stromal stem cell, pIL6 interleukin-6 promoter, TRAIL tumor necrosis factor related apoptosis inducing ligand, UC umbilical cord, WT wildtype