Fig 1.
Morphology of cells cultured in FBS, NBCS, and CS.
Cells were detached with trypsin (0.05%)-EDTA or Accutase and cultured in medium containing fetal bovine serum (FBS), newborn calf serum (NBCS), and bovine calf serum (CS). Cultured cells were photographed at 18 and 36 h after seeding. (A) TW01, (B) OECM-1, and (C) FaDu cells. Black bar represents 50 μm. NBCS-G: Gibco NBCS; NBCS-H: HyClone NBCS.
Fig 2.
Newborn calf serum did not provide favorable conditions for the growth of head and neck cell lines.
The proliferation of cells cultured in NBCS and CS at 72 h was presented as the relative proliferation ratio of cells in FBS (1.0-fold). Data indicate the average value of triplicates (mean ± SD). *:p<0.001 compared with cells cultured in FBS. NBCS-G: Gibco NBCS; NBCS-H: HyClone NBCS.
Fig 3.
Long-term proliferation comparison of cells in FBS and alternative sera.
The proliferation profiles of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, 20, and 30. Cells cultured in FBS were adjusted as the baseline (1.0-fold), and the relative proliferation ratio of cells in other sera was determined accordingly. Data indicate the average value of triplicates (mean ± SD). *:p<0.01 compared with cells cultured in FBS at the same passage. N.D.: not determined for OECM-1 cells cultured in CS due to subculture termination at passage 10.
Fig 4.
Morphology analysis of TW01 cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.
Fig 5.
Morphology analysis of HONE-1 cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.
Fig 6.
Morphology analysis of OECM-1 cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm. N.D.: not determined for OECM-1 cells cultured in CS due to subculture termination at passage 10.
Fig 7.
Morphology analysis of FaDu cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.
Fig 8.
Morphology analysis of SCC25 cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.
Fig 9.
Morphology analysis of DOK cells cultured in alternative sera.
The morphology of cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) were presented at passages 1, 10, and 30. Pictures were obtained at 200× magnification. Black bar represents 50 μm.
Fig 10.
Plating efficiency of cells cultured in alternative sera.
Plating efficiency was determined in cells cultured in fetal bovine serum (FBS), calf serum (CS), iron-supplemented calf serum (ICS), FetalClone III (FC3), Cosmic calf serum (CCS), and Fetalgro (FG) serum. Data indicate the average value of duplicates (mean ± SD). *:p<0.05 compared with cells cultured in FBS. N.D.: not determined because subculture terminated at P10 in OECM-1 cells.
Fig 11.
Migration and invasion of cells cultured in alternative sera.
Migration of cells cultured in FBS and alternative sera at indicated times. The red circle represents the cell front at t = 0 h when the stopper was removed. Yellow bar represents 200 μm. (A) TW01, (B) HONE-1, and (C) FaDu cells. (D) The invasiveness of TW01 cells was determined by the number of cells that invaded and transmigrated to the lower surface of the transwell membrane at 48 h. Data indicate the average value of three wells (migration) or three culture inserts (invasion) (mean ± SD). *:p<0.05; **:p<0.01; and ***:p<0.001 compared with cells cultured in FBS.
Fig 12.
Anchorage-independent growth of cells cultured in alternative sera.
Soft-agar colony formation assays were performed in TW01 and HONE-1 cells. Colony numbers were counted after three weeks of growth. Data indicate the average value of triplicates (mean ± SD). *:p = 0.004 compared with cells cultured in FBS.
Fig 13.
Cytotoxicity assays of cells cultured in alternative sera.
Viability assays of cisplatin-treated cells cultured in different sera were performed using a standard MTT assay at 72 h. Cell viability is presented as a relative value when the mock-treated cells were adjusted to 1.0. (A) TW01 cells. Δ: p<0.05 compared with 10 μM cisplatin-treated cells in FBS. *: p<0.05 compared with 20 μM cisplatin-treated cells in FBS. (B) HONE-1 cells. *: p<0.05 compared with 10 μM cisplatin-treated cells in FBS. (C) FaDu cells. Δ: p<0.05 compared with 5 μM cisplatin-treated cells in FBS. *: p<0.05 compared with 10 μM cisplatin-treated cells in FBS. Data indicate the average value of triplicates (mean ± SD).
Fig 14.
The transcription factor profile of TW01 cells cultured in FBS and FC3.
(A) The scatter plots of the transcription factor profile in TW01 P1 cells. (B) The scatter plots of the transcription factor profile in TW01 P30 cells. The central line indicates unchanged gene expression; dotted lines represent the two-fold regulation cut-off. Genes with fold change > 2 indicated. (C) Western blot analysis of CTNNB1, ELK1, SMAD4, and c-Fos expression in TW01 and HONE-1 P30 cells cultured in different sera. GAPDH is detected as a loading control.