Fig 1: (A) Morphology of cen3 primary fibroblasts and cen3tel cells at different stages of propagation (around PDs 30, 100, 160, 600 and 1000) plated in non-adherent culture conditions, in serum free medium supplemented with EGF and FGFb. Cells grown for 7 days in sphere-forming conditions are shown in the pictures taken with a 10X objective. Bars = 200 µm. (B) Frequencies of primary, secondary and tertiary spheres from cen3tel cells at different PDs. Frequencies of cen3tel cells at PD 100 and 160 were measured 14 days after cell seeding, while those of cen3tel cells at PD 600 and PD 1000 after 7 days. Mean and standard deviation (error bars) values were calculated from three independent experiments. (C) RT-qPCR analysis of SOX2 expression in cen3tel 600 and 1000 sphere cells. SOX2 expression in each sphere sample is expressed as fold change (FC) relative to the expression in the corresponding adherent cells. The plot shows the average (FC) of three independent experiments. (D) Cytofluorimetric analysis of Sox2 expression showing the percentage of Sox2 positive cells in cen3tel 600 and 1000 sphere cells and adherent cells. Values are the average of the results of three independent experiments. Error bars: standard deviations. ***p < 0.005, *p < 0.05. (E,F) Tumor induction by adherent and sphere cells in NSG mice. (E) Tumor growth; (F) percentage of tumors found at the first time of detection (26 days for cen3tel 600 cells and 21 days for cen3tel 1000. Cen3tel 600 (I), first experiment, 4 inoculi; cen3tel 600 (II), second experiment, 8 inoculi; cen3tel 1000, 8 inoculi).
Fig 2: mRNA expression of pluripotency markers in uterine cells isolated from control cows and from cows with adenomyosis. NANOG a OCT4 b and SOX2 c mRNA expression in uterine stromal cells. NANOG d OCT4 e and SOX2 f mRNA expression in uterine myometrial cells. Data were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM. Asterisks indicate statistical differences between uterine normal and adenomyotic tissue (*P < 0.05; **P < 0.01), as determined by Student’s t-test. C – cells obtained from control cows, ADENO – cells obtained from cows with adenomyosis
Fig 3: Protein expression of pluripotency markers in uterine cells isolated from control cows and from cows with adenomyosis. NANOG a OCT4 b and SOX2 c protein expression in uterine stromal cells. NANOG d OCT4 e and SOX2 f protein expression in uterine myometrial cells. Data were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM. Asterisks indicate statistical differences between uterine normal and adenomyotic tissue (*P < 0.05; **P < 0.01), as determined by Student’s t-test. Representative blots for NANOG, OCT4, SOX2 and GAPDH are shown below the graphs g. MM – molecular weight marker, C – cells obtained from control cows, ADENO – cells obtained from cows with adenomyosis
Fig 4: mRNA expression of pluripotency markers NANOG a, OCT4 b and SOX2 c in uterine tissues obtained from control cows and from cows with adenomyosis. Data were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM. Asterisks indicate statistical difference between uterine normal and adenomyotic tissues (P < 0.05), as determined by Student’s t-test. C – tissues obtained from control cows, ADENO – tissues obtained from cows with adenomyosis
Fig 5: Protein expression of NANOG a, OCT4 b and SOX2 c in bovine uterine tissues obtained from control cows and from cows with adenomyosis. Data were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent the mean ± SEM. There were no statistical differences between uterine normal and adenomyotic tissues (P > 0.05), as determined by Student’s t-test. Representative blots for NANOG, OCT4, SOX2 and GAPDH are shown below the graphs d. MM – molecular weight marker, C – tissues obtained from control cows, ADENO – tissues obtained from cows with adenomyosis
Supplier Page from MilliporeSigma for Anti-SOX2 antibody produced in rabbit