Induction of iNOS in MSCs. Total RNA and cell lysates were collected from MSCs alone, splenocytes alone, MSCs cocultured with activated T cells, or activated splenocytes cocultured with MSCs. MSCs were harvested just after washing out activated T cells with PBS. (A) RT-PCR analysis of iNOS mRNA. β-actin is shown as a control. (B) Western blot analysis of iNOS protein. Each lane contains 20 μg protein. β-actin is shown as a loading control. HeLa cells were used as negative control because the antibody also reacts with human iNOS protein. (C) Immunofluorescence of iNOS protein. Left panel, confocal immunofluorescent image of CD45 protein. Middle panel, confocal immunofluorescent image of iNOS protein. Right panel, merged confocal immunofluorescent images of CD45 protein and iNOS protein. Splenocytes (1 × 106) were activated with Con A in the presence of 1 × 105 MSCs for 48 hours. Images were visualized using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) equipped with a 100×/1.40 numerical aperture oil objective lens, Nikon CFI Plan APO (Nikon). Images were acquired using Lasersharp software version 2.1 (Bio-Rad).
