Fig. 5

Pimecrolimus downregulates the COX2-PGE₂ axis in hUCB-MSCs by inhibiting nuclear localization of NFAT3. a The mRNA expression of NFAT isoforms and FKBP1 was determined using conventional PCR. hPBMCs or T cells were used as positive controls. b The expression of the NFAT3 in hUCB-MSCs was verified by immunostaining, Bar = 200 μm. Cells were treated with pimecrolimus (100 ng/ml). c–e Nuclear (c) and Cytosolic (d) fractions were analyzed using Western blotting. hUCB-MSCs were treated with the indicated concentration of pimecrolimus for 3 days. The specificity of the nuclear and cytosolic extracts was determined by performing Western blot analysis with lamin A/C (nuclear) and α-tubulin (cytoplasmic) antibodies. Quantification of NFAT3 and cytosolic COX2 was performed by Image J, and normalized by lamin A/C (nuclear fraction) or α-tubulin (cytosolic fraction). The graph e represents the relative expression level of NFAT3 in nucleus and cytosol of hUCB-MSCs dependent on pimecrolimus treatment. f The expression of NFAT3 and COX2 in hUCB-MSCs. Cells were treated with ionomycin (1 μg/ml) and PMA (100 ng/ml) for 72 h. Bar = 200 μm. g Western blot analysis of hUCB-MSCs revealed COX2 expression and NFAT phosphorylation. Cells were treated with ionomycin (1 μg/ml) and PMA (100 ng/ml) for 72 h with or without pimecrolimus (100 ng/ml). Quantification of COX2 and phosphorylated NFAT3 was performed by imageJ. Expression of COX2 was normalized against GAPDH housekeeping gene, and expression of phosphorylated NFAT3 was normalized against NFAT3 and GAPDH housekeeping gene. The Kruskal–Wallis test with Dunn’s post hoc was used in (e). *P < 0.05 and **P < 0.01. In vitro experiments were performed in triplicate using hUCB-MSCs isolated from each donor (N = 3)