Fig. 7: PHD2 silencing enhances BM-MSCs paracrine effect on NEC via the NF-κB pathway.

a Identification of cytokines of PHDMSC-CM that are potentially beneficial to intestinal recovery. Representative images of the cytokine antibody array are shown. The rectangles highlight cytokines higher in PHDMSC-CM than MSC-CM (fold change > 2.0). Each sample consisted of a pool of unconcentrated conditioned medium from five different donor cells which were derived and sampled independently. Each measurement was duplicated reproducibly. b The graphs show the relative intensity fold changes of the 36 cytokines detected by antibody-based protein array. c Unconcentrated conditioned medium was assayed by ELISA for six selected cytokines. N = 6 independent samples. *P < 0.05 vs. MSC-CM. d The effect of neutralization of selected cytokines or addition of selected exogenous cytokines on apoptosis (Left panel) and proliferation (Middle panel) of IEC-6. The apoptosis and proliferation were evaluated by quantification of TUNEL-positive and PCNA-positive IEC-6 cells in vitro 3 days after exposure, respectively. N = 6. **P < 0.05 vs. NEC + PHDMSC-CM. ^##P < 0.05 vs. NEC + DMEM-F12. (Right panel) The effect of neutralization of selected cytokines or addition of selected exogenous cytokines on survival of NEC rat pups during the first 7 days of life. The cumulated number of rats in each experimental group is presented in parenthesis. **P < 0.05 vs. NEC + DMEM-F12. ^##P < 0.05 vs. NEC + PHDMSC-CM + IgG. e (Upper panel) Representative blots of Western blotting analysis of HIF-1α protein expression in BM-MSCs with or without PHD2 silencing. (Lower panel) Representative blots for phospho-IKKΒ, total IKKα, total IKKΒ, phospho-IκBα, and total IκBα in BM-MSCs with or without PHD2 silencing. β-actin is used as control. f Effect of PHD2 silencing on nuclear translocation of NF-κB in BM-MSCs. Scale bars 10 μm. g The DNA-binding activities of NF-κB in BM-MSCs with or without PHD2 silencing were estimated by electrophoretic mobility shift assay (EMSA). The specificity of the DNA/protein was determined by competition reactions in which a 100-fold molar excess of unlabeled NF-κB oligonucleotide (specific competitor). h The effect of HIF-1α silencing and NF-κB inhibition on pivotal cytokines secretion of BM-MSCs were determined by ELISA. N = 6 independent samples. *P < 0.05 vs. PHDMSC-CM. i The effect of NF-κB inhibition on BM-MSC paracrine mediated effect on intestinal epithelial cells in rats with NEC. NF-κB inhibition but not HIF-1α silencing blocked the therapeutic potential of PHDMSC-CM to ameliorate the proliferation (left panel) and apoptosis (right panel) of intestinal epithelial cells in rats with NEC which were evaluated by quantification of TUNEL-positive and PCNA-positive cells in histological sections, respectively. **P < 0.05 vs. NEC + DMEM-F12. ^##P < 0.05 vs. NEC + PHDMSC-CM. j Improved survival of rats with NEC receiving PHDMSC-CM was reversed by NF-κB inhibition. The cumulated number of rats in each experimental group is presented in parenthesis. **P < 0.05 vs. NEC + DMEM-F12. ^##P < 0.05 vs. NEC + PHDMSC-CM. k Schematic diagram of primer pairs in ChIP analysis. (Upper panel) The rIGF-1 promoter region contains four NF-κB-binding sites. Three primer pairs were designed to detect regions a, b, and c in ChIP assays. (Lower panel) The rTGF-β2 promoter region contains two NF-κB-binding sites. Two primer pairs were designed to detect regions a and b in ChIP assays. l The p50 bound to the κB site within the promoter of rIGF-1 and rTGF-β2 detected by ChIP assay. ChIP analysis was performed using anti-p65, anti-p50, or control IgG antibodies for immunoprecipitation followed by PCR using primers for the specific rIGF-1/rTGF-β2 promoter region in which the predicted NF-κB site is located. The PCR products were subjected to electrophoresis on 2% agarose gel. These results are representative of three independent experiments.