Fig 2: The supernatants derived from breast cancer cells four days after drug withdrawal enhance CSC phenotypes and chemoresistance. (a) Experimental design for the study of autocrine factors in supernatants derived from chemotherapy-treated breast cancer cells. SUM190, SUM149 and MDA-MB-231 cells were treated with 15 nM paclitaxel for 4 days. After medium change and wash with PBS, the cells were cultured in fresh medium in the absence of any drug for additional 4 days. At day 8, the cell-free and drug-free culture media (Supernatants) were collected and applied to the same line of fresh cells respectively for 4 days. (b) Flow cytometric analysis of the percentage of CD44high/CD24-/low cells in SUM190, SUM149 and MDA-MB-231 cells after exposure to the supernatants for 4 days. The 7-AAD-negative live cells were pre-gated. Data represent means±S.D., n=3; * P<0.05. Veh-SNs: supernatants (SNs) derived from vehicle (Veh)-pretreated cells; Pa-SNs: supernatants derived from 15 nM paclitaxel (Pa)-pretreated cells. (c) Western blot analysis of stem cell-like markers (ALDH1, CD44 and OCT4) in SUM149 and SUM190 cells after exposure to the supernatants for 4 days. Supernatants (SNs) were collected as described in a and b. Pa 5 nM-SNs or Pa 15 nM-SNs: pretreated with 5 or 15 nM paclitaxel. β-actin was used as an internal loading control. (d) Representative images from mammosphere assays. SUM190, SUM149 and MDA-MB-231 breast cancer cells were treated for 4 days with vehicle (Veh)-derived or paclitaxel (Pa)-derived supernatants (SNs), and then reseeded in ultra-low attachment plate (2 × 103/well, 6-well plate) and cultured for 8 days. Scale bar, 100 μm. Data represent means±S.D., n=3; *P<0.05. (e)qPCR analysis of stemness-associated (ALDH1, SOX2, OCT4, NANOG, CD44 and c-MYC), epithelial-to-mesenchymal transition (EMT)- (SLUG, MMP9, ZEB1, SNAIL and E-CADHERIN) signature genes in SUM190, SUM149 and MDA-MB-231 cells after exposure to the supernatants (SNs) for 4 days. Data represent means±S.D., n=3; *P<0.05 compared with vehicle-supernatants group. See also Supplementary Figure 1

Fig 3: Evaluation of germ cell composition in fresh, vitrified/warmed (warming), or vitrified/warmed/cultured (cultured 24 h) testicular tissue from black-footed ferrets. (A) Proportion of cell stained with Oct4 (pre-meiotic stage), (B) Boule (meiotic stage). Data are expressed as mean ± SE (n = 5 animals per treatment). Different letters above bars indicate significant statistical differences between treatments (p < 0.05).

Fig 4: SATB2 knockdown suppresses hypoxia-induced stemness of SCC9 cells. (A) The relative protein expression levels of Oct-4, Sox-2 and Naong were examined by western blot analysis in SCC9 cells exposed to normoxic or hypoxic conditions for the indicated times. Protein levels were normalized by comparison with β-actin levels. **P<0.01, ***P<0.001 vs. normoxia group. (B) Relative protein expression levels of Oct-4, Sox-2 and Nanog were detected by western blot analysis in SCC9 cells transfected with either si-NC or si-SATB2-2 and cultured under hypoxic conditions for 24 h. Protein levels were normalized by comparison with β-actin levels. ***P<0.001 vs. hypoxia control group. (C) Images of colonies stained with crystal violet, and quantification of the number of colonies formed in SCC9 cells transfected with either si-NC or si-SATB2-2 and cultured under hypoxic conditions for 12 days. **P<0.01 vs. hypoxia control group. (D) The relative protein expression levels of Oct-4, Sox-2 and Nanog were detected by western blot analysis in SCC9 cells transfected with either empty (pcDNA3.1) or SATB2-overexpression plasmids and cultured under normoxic conditions. **P<0.01, ***P<0.001 vs. normoxia group. The results are presented as the mean ± SD of three independent experiments. SATB2, special AT-rich sequence-binding protein 2; si, small interfering; NC, negative control; norm, normoxia; hypo, hypoxic; ctrl, control.

Fig 5: Characterization of adult porcine dermal fibroblasts. (A) Fibroblasts grew out of dermal tissue fragments within 6 days (left panel) and formed a monolayer (right panel). Cells displayed a standard morphology elongated in shape (right panel). Scale bars: 100 μm. (B) Isolated fibroblasts showed a uniform immuno-positivity for vimentin (VIM) and the complete absence of the pluripotent- (OCT4 and NANOG) and TR- (CDX2, KRT7, HSD17B1 and INFG) related markers. Nuclei were stained with DAPI (Scale bars: 100 μm).