Table 1.
Summary of the effect of vitamin C treatment on the gene expression of primary mouse embryonic fibroblasts as determined by gene expression microarray analysisa.
Table 2.
Summary of the effect of immortalization on the gene expression of mouse embryonic fibroblasts as determined by gene expression microarray analysisa.
Table 3.
Summary of the effect of vitamin C treatment on the gene expression of immortalized mouse embryonic fibroblasts as determined by gene expression microarray analysisa.
Figure 1.
Dose- and treatment duration-dependent effect of vitamin C and its analogs on primary mouse embryonic fibroblasts.
Cells propagated in normal medium at low passages (before passage 6 except 1E and 1F) were used for the study. They were seeded in cell culture wells at 2×104 cells/cm2 and treated every day starting the day after seeding. They were harvested 24 hours after the last treatment. There was no medium change from cell seeding to the end of ascorbate 2-phopshate treatment. The medium was changed before the start of the thymidine incorporation experiment as a part of the experimental procedure. (A) Effect of 1–4 days of treatment with 5–20 µM ascorbate 2-phosphate on total protein; (B) Effect of 1–2 days of treatment with 5–20 µM ascorbate 2-phosphate on thymidine incorporation; (C)(D) Passage-dependent effect of ascorbate 2-phosphate on mouse embryonic fibroblasts propogated in the absence (C) or presence (D) of 20 µM ascorbate 2-phosphate; (E) (F) Effect of 4-day treatment of 20 µM ascorbic acid (E) or 20 µM isoascorbate (F) on total protein; (G–I) Effect of 4-day treatment of (G) 100 µM vitamin C or analog, (H) 0.01–1 µM (-)-epigallocatechin-3-gallate (EGCG), or (I) 1–10 µM alpha-tocopherol succinate (Vit E) on total protein. All data shown represent means±S.D. of 3–4 independent wells. *significantly different from the control cells without treatment at p<0.05.
Figure 2.
Confocal immunofluorescence visualization of collagen IV and ß-actin expression in primary embryonic fibroblasts with (bottom row) and without (top row) 4-day ascorbate 2-phosphate treatment.
There was no medium change between cell seeding and harvesting for staining. Composite confocal images of cells stained for collagen IV (second column) and F-actin (third column) were merged (first column) and images of X-Z sections were captured. The size of the merged X-Y image was 140 µm×140 µm. The X-Z section was taken from a stack of thirty-five 0.46 µm slices.
Figure 3.
Vitamin C in the medium does not affect SVCT2−/− embryonic fibroblasts.
(A) SVCT2−/− fibroblasts do not have sodium-dependent vitamin C transport activity. ND: determined but not detected. (B,C) SVCT2−/− fibroblasts do not show vitamin C treatment-dependent increase in (B) total protein (measured after 4-day treatment with 20 µM ascorbate 2-phosphate) or (C) thymidine incorporation (measured after 2-day treatment with 20 µM ascorbate 2-phosphate). There was no medium change from cell seeding to the end of ascorbate 2-phopshate treatment. The medium was changed before the start of the thymidine incorporation experiment as a part of the experimental procedure. Unlike the wildtype fibroblasts, SVCT2−/− fibroblasts did not show (D) passage-dependent; or (E) dose-dependent response to ascorbate 2-phosphate in the medium; but did display (F) dose-dependent response to vitamin E in the medium. All data shown represent means±S.D. of 3–4 independent wells. *significantly different from control cells without treatment at p<0.05.
Figure 4.
Confocal immunofluorescence visualization of collagen IV and ß-actin expression in primary SVCT2−/− embryonic fibroblasts with (bottom row) and without (top row) 4-day ascorbate 2-phosphate treatment.
There was no medium change between the cell seeding and harvesting for staining. Composite confocal images of cells stained for collagen IV (second column) and F-actin (third column) were merged (first column) and images of X-Z section were captured. The size of the merged X-Y image was 140 µm×140 µm. The X-Z section was from a stack of thirty-five 0.46 µm slices.
Figure 5.
The immortalization of wildtype and SVCT2−/− fibroblasts.
Wildtype and SVCT2−/− mouse embryonic fibroblasts were carried to beyond immortalization following the published limited medium method [20]. The immortalization process was associated with (A) a significant increase in days to reach confluence around passage 11. Once immortalized, MEF regained rapid cell division. (B) Saturation density was higher in both wildtype and SVCT2−/− primary fibroblasts compared to their respective immortalized fibroblasts.
Figure 6.
The effect of vitamin C treatment on immortalized wildtype and SVCT2−/− fibroblasts.
(A) The effect of 4-day 20 µM ascorbate 2-phosphate treatment on the total protein of immortalized wildtype fibroblasts. (B,C) Dose-dependent effect of 4-day treatment with (B) ascorbte 2-phosphate and (C) ascorbate on the immortalized wildtype fibroblasts. (D) Immortalized SVCT2−/− MEF did not respond to 4-day 20 µM ascorbate 2-phosphate treatment. (E) Immortalized SVCT2−/− fibroblasts were inhibited only by 4-day 100 µM isoascorbate treatment. (F) Immortalized wildtype fibroblasts were more sensitive to the inhibitory effect of pharmacological concentrations of vitamin C and analog compared to immortalized SVCT2−/− fibroblasts (4-day treatment). All data shown represent means±S.D. of 3–4 independent wells. Some values and error bars are too small to be clearly visible on the graph. *significantly different from the control cells without treatment at p<0.05.
Table 4.
Effect of vitamin C treatment on the 20 genes that had maximal increase in expression upon immortalization in mouse embryonic fibroblasts (MEF).