Figure 2.

NF-κB activation in the apoptotic MSCs produces an immunosuppressive secretome independent of the dying process

(A) To knock down the RelA in the bone marrow MSCs, MSCs were infected by lentivirus vector using pLentiLox.3.7 with the shRNA control sequence (Sh-Ctl) and RelA sequence (Sh-RelA) for 48 h at 37°C, 5% CO₂. Expression of RelA (65 kDa) was assessed by immunoblotting. β-Tubulin (43 kDa) was used as the loading control. (B) After 24-h treatment of anti-Fas, the percentage of apoptosis in the MSCs was assessed using Annexin-V and 7AAD assay. (C) The intracellular COX2 level was assessed using flow cytometry as described before. (D–F and H) The level of PGE2, LIF, and IL-6 in the cell culture supernatant were assessed using human PGE2, LIF, and IL-6 ELISA kits, respectively. One-way ANOVA and post hoc Tukey test were used to compare the mean differences among the samples. (G) Dot plots illustrating the distribution of COX2 expression among the Annexin-V⁺ or Annexin-V⁻ population. Thirty micromolar BAY 11–7085 was added to MSCs during the Fas stimulation to prevent NF-κB activation. Two-way ANOVA and post hoc Tukey test were used to compared the mean differences between groups. ∗ p values < 0.05; ∗∗,^## p values < 0.01; ∗∗∗, ˆˆˆ p values < 0.001; ∗∗∗∗, ^####, ˆˆˆˆ p values <0.0001; n.s, no significance. All results shown are representative of at least three independent experiments. Experimental data were expressed as mean ± SD.