Fig 2: Macrophage migration inhibitory factor (MIF) promotes CD74-dependent cardiac stem cell (CSC) proliferation. (A and B) CSCs were transfected with a specific siRNA against CD74 (siRNA-CD74) or transfected with a negative control siRNA (siRNA-NC). Knockdown efficiency was examined by western blot analysis. (C and D) CSCs in which CD74 was knocked down were treated with MIF (200 ng/ml) for 48 h. Flow cytometric analysis was used to detect the cell cycle, and the percentage of cells in the S phase decreased in the siRNA-CD74 group compared with the siRNA-NC group. (E and F) siRNA-transfected CSCs were treated with MIF (200 ng/ml) for 24 h and were cultured for additional 2 h in medium with 50 µM EdU. Cells were fixed, permeabilized and stained with the Apollo® reaction cocktail and Hoechst 33342. Data in (B, D and F) are the means ± SD of 3 independent experiments (n=3). **p<0.01 vs. control or siRNA-NC.

Fig 3: Macrophage migration inhibitory factor (MIF) activates the PI3K/Akt/mTOR pathway and adenosine monophosphate-activated protein kinase (AMPK) in cardiac stem cells (CSCs). (A–C) Following treatment wiht MIF (200 ng/ml) for various periods of time, the cells were harvested and western blot analyses were performed to compare the changes in the levels of (A) phospho-Akt (T308), (B) phospho-mTOR(S2448), and (C) phospho-AMPK(T172). (D–E) CSCs were pre-treated with LY294002 (PI3K inhibitor, 25 mM) or MK-2206 (Akt inhibitor, 20 mM) for 90 min, or transfected with siRNA-NC or siRNA-CD74 for 72 h. MIF (200 ng/ml) was then added in the presence of each drug followed by culture for an additional 1 h. (D) Akt and phospho-Akt (T308), (E) mTOR and phospho-mTOR (S2448), and (F) AMPK and phospho-AMPK (T172). Data are the means ± SD of 3 independent experiments (n=3). *p<0.05 and **p<0.01 vs. control.

Fig 4: Macrophage migration inhibitory factor (MIF) promotes cardiac stem cell (CSC) proliferation. (A) MIF affects CSC viability in a dose-dependent manner. Cells were cultured with various concentrations of MIF in 96-well plates at a density of 5×103 cells/well. After 1 or 2 days in culture, cell viability was measured by CCK-8 assay. (B and C) Flow cytometric analysis of CSCs cultured in the presence of MIF (200 ng/ml) or ISO-1 (100 µg/ml). After 2 days, the percentage of cells in the S-phase was increased in the MIF treatment group compared to the control, and decreased in the ISO-1 treatment group. (D and E) MIF treatment increased EdU incorporation by CSCs. Cells were treated with MIF (200 ng/ml) or ISO-1 (100 µg/ml) for 1 day and then cultured for 2 h in medium with 50 µM EdU. Cells were fixed, permeabilized and stained with the Apollo® reaction cocktail and Hoechst 33342. Data in (A, C and E) are the means ± SD of 3 independent experiments (n=3). **p<0.01 vs. control, #p<0.01 indicates MIF + ISO-1 vs. MIF.

Fig 5: Macrophage migration inhibitory factor (MIF) and CD74 expression in cardiac stem cells (CSCs). (A and B) MIF and CD74 PCR products run on a 2% agarose gel. Thymus, liver and bone marrow-derived mesenchymal stem cells (BM-MSCs) were used as positive controls. (C) CD74 immunoreactivity (green) in cultured mouse CSCs. The merged image is also shown. (D) CD74 mRNA expression was analyzed by qRT-PCR before and after MIF stimulation. There was no statistical significance. Data are the means ± SD of 3 independent experiments (n=3).