Figure 4

Effect of gamma-secretase on IL-1 signalling and PGE2 content. A: UC-MSCs were cultured in presence of different doses of IL-1β stimulation with or without DAPT (gamma-secretase inhibitor). UC-MSCs were detached using accutase and stained to analyse intracellular COX-2 expression (n = 5). B: Gamma-secretase activity in UC-MSCs was silenced using siRNA targeting a catalytic subunit of gamma-secretase (PSEN-1). Scrambled siRNA (SCR) was also introduced into UC-MSCs as control. After 24 hours, PSEN-1 or SCR siRNA treated MSCs were stimulated with IL-1β overnight. UC-MSCs were detached using accutase and stained to compare intracellular COX-2 expression with or without IL-1β stimulation (n = 3). C: UC-MSCs were stimulated with IL-1β for 40 minutes, with or without 2.5 hours pre-treatment with DAPT. MSCs were detached using accutase, immediately fixed, permeabilised and stained to analyse intracellular pJNK levels (n = 4). D: NK cells were cultured with or without MSCs or co-cultured in presence of 6 μM DAPT, 10 μM DAPT or DMSO. CD107a degranulation assay was performed with K562 target cells. The bars represent the effect of gamma-secretase inhibition on degranulative capacity of NK cells (n = 5). E: NK cells were cultured in NK cell conditioned media (NK cm) or in NK-MSC conditioned media (NK-MSC cm) or NK-MSC conditioned media where they were co-cultured in presence of 10 μM DAPT or DMSO (NK-MSC-10 μM DAPT cm or NK-MSC-DMSO cm respectively). Chromium release assay was performed with K562 target cells (n = 3). F: PGE2 concentration in MSC cm, NK-MSC cm or NK-MSC-10 μM DAPT cm as determined by competitive ELISA (n = 3).