Fig 2: Increase in IDO level in hASC in the presence of activated PBMC. Supernatants from mixed cultures of activated PBMC (anti-CD3, anti-CD28) cultured with hASC (48 h) affect the CD25 level in CD4 T cells. After 48 hours of incubation, PBMC were analyzed for CD25 (IL-2Ra) surface expression (n = 3, mean + SEM) (a). Changes in IDO transcription after treatment of hASC with supernantants (n = 3) (b). Transcriptional activity of IDO (c) and iNOS (d) genes in the course of hASC-mediated immune suppression (n = 4). IDO protein level in hASC cultured with anti-CD3- and anti-CD28-stimulated PBMC at 1 : 25 ratio for 48 hours (vinculin-normalization control) (e) (*p < 0.05 and **p < 0.01; unstim—unstimulated PBMC, stim—PBMC stimulated with anti-CD3 and anti-CD28).

Fig 3: Immunohistochemical staining of the newly formed bone one week after implantation. (A) In the Defect and PBS groups, CD204-positive cells gradually increased compared with the specimens after 72 h. In the MSC-CM group, fewer iNos-positive cells and many CD204-positive cells were observed around the bone edge. Bars = 200 µm (B) The number of iNOS-positive cells was significantly reduced in the MSC-CM group 1 week after implantation, but the number of CD204-positive cells in the MSC-CM group was relatively decreased 1 week after implantation.

Fig 4: (S)-(+)-carvone (4) and (R)-(-)-carvone (5) decrease LPS-induced Nos2 mRNA (a) and protein (b) levels in the Raw 264.7 cell line. Macrophage cultures were treated with 1 µg/mL LPS, for 6 h (a) or 18 h (b), following pre-treatment for 1 h with 666 µM of each test compound (a) or with the concentrations indicated in (b). As a positive control, the cells were similarly treated with the selective NF-?B inhibitor, Bay 11-7082, 5 µM. Control cells (Ctrl) were treated with the vehicle (0.1% DMSO) in the absence of LPS. Each column represents the mean ± SEM of four independent experiments. The blots shown are representative of, at least, three independent experiments and are cropped for clarity and conciseness. The corresponding full-length blots are presented in Fig. S3. **p < 0.01, ***p < 0.001 and ****p < 0.0001 relative to LPS-treated cells. ##p < 0.01 relative to the Ctrl. §p < 0.05, §§§p < 0.001 and §§§§p < 0.0001 between the conditions indicated. MW: molecular weight marker.

Fig 5: Immunohistochemical staining of the newly formed bone 72 h after implantation. (A) Hematoxylin and eosin (H–E) staining indicates the orientation of the specimens, showing the bone edge of the defect where bone formation will start. In the Defect and PBS groups, there were many iNOS-positive cells and fewer CD204-positive cells around the bone edge. However, in the MSC-CM group, fewer iNOS-positive cells and numerous CD204-positive cells were observed around the bone edge. Bars = 200 µm (B) The number of iNOS-positive cells was significantly reduced in the MSC-CM group 72 h after implantation, and the number of CD204-positive cells in the MSC-CM group was significantly increased after 72 h after implantation.