Fig 1.
An overview of the experimental procedures that have been done in this paper.
Table 1.
Primer sequences used for RT and Real time-PCR.
Fig 2.
Morphological features of bone marrow derived-MSCs; (A) Spindle-shaped morphology of bone marrow cells that appear at day 1, (B) The number and size of colony appears to gradually increase on days 3–7, (C) More confluent bone marrow derived-MSCs at 21 days of culturing cells. (D) population doubling time (PDT) of bone marrow derived-MSCs. Two-lineage differentiation of bone marrow derived-MSCs; (E) Generation of lipid vacuoles after adipogenesis and staining by Sudan III; expression of PPAR-α and PPAR-γ as fat-specific genes, (F) Osteogenic differentiation and Alizarin red staining of mineralized cell aggregates; detection of the two bone specific genes (ALP and Runx2) by RT-PCR method. The undifferentiated cells were used as control in RT-PCR (scale bar = 40X).
Fig 3.
The expression of the cell surface markers of bone marrow derived-MSCs that analyzed by flow cytometry.
Each cell surface marker was tested individually by separate cell population distributions and the isotopes controls were used as the negative control in this experiment; (A) a total population of cells for CD90 evaluation, (B) the bone marrow derived-MSCs were positive for CD90 (90.8%), (C) a total population of cells for CD44 evaluation, (D) the bone marrow derived-MSCs were positive for CD44 (84.6%), (E) a total population of cells for CD34 evaluation, (F) the bone marrow derived-MSCs were negative for CD34 (0.57%), (G) a total population of cells for CD56 evaluation, (H) the bone marrow derived-MSCs were negative for CD56 (0.23%). For CD34 and CD56, the isotype control was mouse IgG1 and for CD90 and CD44, the isotype control was mouse IgG2b. Also, isotype control is seen with red dots.
Fig 4.
Antibody array analyses of cytokines present in bone marrow derived-MSCs.
(A) Cytokines presence in culture medium of bone marrow derived-MSCs alone, (B) Cytokines presence in culture medium of K562 alone, (C) Cytokines presence in culture medium of bone marrow derived-MSCs co-cultured with K562 cells, (D) Quantification of cytokine array; (****P < 0.0001).
Fig 5.
(A-D) Representative image of cell cycle distribution. A and C are cell cycle distribution of K562 cells alone (control group); B and D are cell cycle distribution of K562 cells co-cultured with bone marrow derived-MSCs (experimental group). (E) Quantification analysis of cell cycle distribution; (*P < 0.05 and **P < 0.01 compared with control group).
Fig 6.
Flow cytometric analysis of bone marrow derived-MSCs co-cultured with K562 cells was performed with a combination of Annexin V-FITC, propidium iodide (PI).
A shift from bottom-right quadrant panel (early apoptosis) to top-right quadrant panel (late apoptosis) and top-left quadrant panel (necrosis) was observed. (A) Unstained cells, (B) Control group and (C) Experimental group.
Fig 7.
Proliferation and apoptosis of K562 cell line following co-culture with bone marrow derived-MSCs.
Harvested cells were evaluated with Ki-67 (A-E) and apoptosis (F-J) by flow cytometry. In this figure, A and F are selected cell population, B and G are isotype control, C and H are K562 cells alone, finally D and I are K562 cells co-cultured with bone marrow derived-MSCs. Values are mean ± SD from independent experiments; (**p<0.01).
Fig 8.
Effect of bone marrow derived-MSCs on apoptosis related gene and protein expression.
(A) The mRNA and (C and D) the protein expression levels of BAX, BCL-2. (B) Bax/Bcl-2 ratio. The data presented as mean±SD of 3 independent experiments. *p< 0.05; ***p< 0.001 and ****p< 0.0001.