Figure 1.

Fas stimulation induces the non-death signaling in bone marrow MSCs during apoptosis

(A) Bone marrow MSCs from different batches (5 × 10⁵ per 96 well) were treated with 5 μg/mL recombinant granzyme B and 10 μg/mL anti-Fas clone CH11 in complete RPMI for 24 h at 37°C, 5% CO₂. A total of 10 samples from five independent groups were prepared for the human microarray analysis. GSEA used the hallmark gene set to analyze differently expressed genes in ApoMSCs compared with MSCs. Other parameters are set as default. Gene sets with FDR less than 0.25 are considered as significant. (B) Clustered heatmap was generated using the heatmap.2 function from R.Studio. Within the gene set of TNF-α signaling via NF-κB, 76 genes were identified as core genes that contributed most to the enrichment result. Within those 76 genes, there is a list of immunomodulatory factors and chemokines. (C) RNA was isolated from untreated MSCs and Fas-stimulated MSCs, which were treated with 10 μg/mL anti-Fas (clone CH-11) for 24 h at 37°C, 5% CO₂. Expression levels of LIF, TNFAIP6, PTGS2, and IL-6 were determined by qRT-PCR. The bar chart showed the relative fold changes of markers in comparison with untreated MSC after normalization with HPRT-1. (D) Increased expression of TSG-6 (35 kDa) and COX2 (70 kDa) in Fas-stimulated MSCs was confirmed also at the protein level by immunoblotting. α-Tubulin (55 kDa) was used as loading control. ImageJ software was used to quantify the intensity of bands. The expression level of TSG-6 and COX2 in each experiment was normalized to the α-Tubulin. (E) ELISA analysis also confirmed the increased level of LIF, IL-6, and PGE2 in the supernatant of Fas-stimulated MSCs compared with the untreated. (D and E) Experimental data were expressed as mean ± SD. Unpaired t test was used to compare the mean difference between the two groups. (F) Dot plots illustrating the distribution of COX2 expression among the Annexin-V⁺ or Annexin-V⁻ population. (G) The intracellular COX2 was stained using human anti-COX2 (clone AS67) and the COX2⁺ population was gated according to the isotype control. 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. (F and G) All results shown are representative of least three independent experiments. One-way ANOVA and post hoc Tukey test were used to compare the mean differences among the samples. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗,### p values <0.001; ∗∗∗∗, ####, ˆˆˆˆ p values <0.0001; n.s, no significance.