Fig 2: Neutralization of G-CSF abolishes the protective effects of hUCMSC-CM on ovaries. (A) Images of sections from P5 mouse ovaries stained with TUNEL (red) after treatment with Cs (2 µg/ml) and hUCMSC-CM in the presence or absence of anti-GCSF for 12 h. White arrowheads indicate the apoptotic primordial follicles. Nuclei were stained with DAPI. Scale bar, 50 µm. (B) Ovary sections used for H&E staining and DDX4 immunofluorescence (cytoplasm, green) were obtained from P9 mice. Anti-GCSF was administered via intraperitoneal injection every other day beginning on P4. Cs (5 mg/kg body weight) was administered via intraperitoneal injection on P5, and hUCMSC-CM was injected every other day beginning on P5. Black arrowheads indicate primordial follicles. Nuclei were stained with DAPI. Scale bar, 20 µm. Images are representative of at least three independent experiments.

Fig 3: Effects of Nano-DOX and DOX on MDSCs infiltration in tissues and G-CSF expression. a Immunofluorescent staining of MDSCs markers CD11b and Gr-1 in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. b Immunofluorescent staining of MDSCs markers CD11b and Gr-1 in spleen and H&E staining of granulocytes in liver and lungs. c RT-PCR analysis of G-CSF mRNA levels in in vitro 4T1 cells treated with Nano-DOX or DOX. d ELISA analysis of G-CSF secretion from in vitro 4T1 cells treated with Nano-DOX or DOX. e Immunohistochemical staining of G-CSF in 4T1 tumor xenografts at the end of 3-week treatment of Nano-DOX or DOX. f Serum levels of G-CSF in 4T1 tumor-bearing mice at the end of 3-week treatment of Nano-DOX or DOX, assayed by ELISA. (Duration of Nano-DOX or DOX treatment was 24 h for the in vitro cell experiments.) Values were mean ± SD (n = 3 for in vitro experiments and n = 5 for in vivo experiments, *p < 0.05, **p < 0.01)

Fig 4: Downregulation of G-CSF increases the apoptosis of granulosa cells. Granulosa cells were transfected for 24 h with G-CSF or control siRNA before treatment with recombinant G-CSF for 24 h. (A) qRT-PCR and western blot results of G-CSF in granulosa cells after G-CSF or control siRNA transfection. (B) Flow cytometry analysis of apoptotic granulosa cells after recombinant G-CSF treatment. (C) PI3K and Akt phosphorylation in granulosa cells after recombinant G-CSF treatment. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05.

Fig 5: Verification of the expression of G-CSF, GM-CSF, Cxcl1, Ccl2, Ccl7, and Il23a in the ovaries of P5 mice treated with Cs (2 µg/ml) alone or in combination with hUCMSC-CM using qRT-PCR. Data are presented as mean ± SD of at least three independent experiments. *P < 0.05.