Fig. 6
Succinate Enhances the Effect of Peritoneal mesothelial cells on Macrophage Recruitment and ESCs Adhesion. A, B RT-PCR was used to analyzed the CCL2 mRNA in HMrSV5 cells treated with vehicle or succinate (0, 1 and 2.5 mM) for 48 h, and ELISA was used to detected protein level of CCL2 (n = 3). All were analyzed by one-way ANOVA followed by Dunnett’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean ± SEM (CCL-2 group was positive control). C hESCs were seeded in upper chamber and HMrSV5 cells were treated with 2.5 mM succinate in lower chamber for 48 h, and then the hESCs was replaced with THP-1 cells for another 12 h. THP-1 cells were prelabeled with red fluorescent reagent CellTracker red. The chemotaxis assay for THP-1 cells was calculated as cell number per field (scale bar-200 μm) (red tracer staining THP-1 cell) (n = 3). One-way ANOVA followed by Dunnett’s post hoc test, * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean ± SEM (CCL-2 group was positive control). D Adhesion assays show hESCs adhere to HMrSV5 cells pretreated with different concentration of succinate for 48 h. hESCs(1 × 105 cells/well) were prelabeled with green fluorescent reagent CellTracker green and seeded in the HMrSV5-well. Results are from 3 independent trials (n ≥ 3 for mimic) and data depicted as column mean graphs with error bars showing confidence intervals (scale bar-100um). E RT-PCR of ICAM-1 in HMrSV5 cells treated with vehicle or succinate (1,2.5 and 5 mM) for 48 h and analyzed by one-way ANOVA followed by Dunnett’s post hoc test (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean ± SEM. F WB of ICAM-1 in HMrSV5 cells treated with different concentrations of succinate for 48 h. G Transwell migration assay of hESC in the presence of HMrSV5 cells with vehicle or 2.5 mM succinate treatment for 48 h. Media with CCL2 100 ng/ml. H RT-PCR of MMP2, MMP9 and MMP11 in HMrSV5 cells treated with succinate (0, 1, 2.5 and 5 mM) for 48 h and analyzed by one-way ANOVA followed by Dunnett’s post hoc test (n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean ± SEM