Fig 1.
Doc reduced the cell viability.
(A) SKOV3 and A2780 cells were treated with different concentrations of Doc (0, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50, 100, 500, 1000 nM) overnight (16–18 h). The cell viability was determined by MTT assay (N = 5). P values were calculated using the student’s t-test. *P<0.05, **P<0.01, ***P<0.001 (vs. 0 nM). (B and C) SKOV3 and A2780 cells were treated with Doc (200, 400,-800 nM) overnight (16–18 h) (B), and then allowed to recover in the regular medium for another three days (C). The morphological evaluations were done by a microscopy and representative image was shown (magnification 10×10). Bar = 100 μm.
Fig 2.
Doc promoted the formation of PGCCs.
(A) The morphology of cells was imaged under a microscope followed by processing with the NIS-Elements D 3.2 software. Representative image was shown (magnification 10×10). Bar = 100 μm. Bar graphs showed the diameter of cells (n = 50). (B) Polyploid phenomena were analyzed by flow cytometry. Representative histogram was shown, and the region marked by black line indicated by P2 showed the polyploid cells (DNA>4N). Bar graphs showed the percentage of polyploid cells (n = 3). P values were calculated by One-way ANOVA. **P<0.01, ***P<0.001 (vs. control); ###P<0.001 (PGCCs vs. Doc). (C) Hochest 33342 staining was used to reveal the morphology of nucleus. Representative image was shown (magnification 10×20). Red arrow indicated multinuclear cell and white arrow indicated micronucleus cell. Bar = 100 μm.
Fig 3.
Doc induced G2/M cell cycle arrest and inhibited proliferation.
(A) The cell cycle distribution was analyzed by flow cytometry followed by processing with the Flow Jo 7.6 software. Representative histogram was shown. Bar graphs showed the percentage of cells in G2/M phase (n = 3). P values were calculated using the student’s t test. ***P<0.001 (vs. control). (B) The number of cells was counted and bar graphs showed the cell density (n = 3). P values were calculated using the One-way ANOVA. ***P<0.001 (vs. control); #P<0.05, ###P<0.001 (PGCCs vs. Doc).
Fig 4.
Doc activated persistent DDR signaling.
The expression of γ-H2A.X was detected by flow cytometry followed by processing with the BD FACSDiva software. Representative histogram was shown, and the region marked by the black line indicated by P2 showed the γ-H2A.X-positive cells. Bar graphs showed the percentage of γ-H2A.X-positive cells (left, n = 3) and the mean fluorescence intensity (MFI) of γ-H2A.X (right, n = 3). P values were calculated by One-way ANOVA. *P<0.05, **P<0.01, ***P<0.001 (vs. control); ##P<0.01, ###P<0.001 (PGCCs vs. Doc).
Fig 5.
The SA-β-Gal activity was increased and the mitochondrial membrane potential was lost in PGCCs.
(A) SA-β-Gal staining to reveal the activity of β-galactosidase. Representative image was shown (magnification 10×20). Black arrows indicated the SA-β-Gal-positive cell. Bar = 100 μm. (B) JC-1 staining to reveal the mitochondrial membrane potential. Representative scatter plot was shown. Bar graphs showed the percentage of JC-1 monomeric form (n = 3). P values were calculated by One-way ANOVA. ***P<0.001 (vs. control).
Fig 6.
The expression of stem cell markers was upregulated in PGCCs.
Relative mRNA level of KLF4 and OCT4 was determined by RT-qPCR. The values were normalized to GAPDH and relative to the control group on the basis of 2-ΔΔCt method. P values were calculated using the student’s t test. **P<0.01 (vs. control).