Uptake of folic acid by preconfluent, confluent, and postconfluent Caco-2 cells. Initial rate of carrier-mediated uptake of [³H]folic acid (9 nM) by preconfluent, confluent, and postconfluent Caco-2 cells. Incubation was performed at 37°C in Krebs-Ringer buffer (pH 5.5). Folic acid uptake was determined as described in materials and methods. Data represent means ± SE of at least 3 independent uptake experiments.

Level of expression of human reduced folate carrier (hRFC) and human proton-coupled folate transporter (hPCFT) protein and mRNA as well as activity of the SLC19A1 and SLC46A1 promoters in preconfluent, confluent, and postconfluent Caco-2 cells. A and D: Western blot analysis was performed using membranous fraction (150 μg) isolated from preconfluent, confluent, and postconfluent Caco-2 cells and specific anti-hRFC and anti-hPCFT polyclonal antibodies. Using an enhanced chemiluminescence kit (Amersham, Arlington Heights, IL), immunoreactive bands were detected as described in materials and methods. B and E: quantitative real-time PCR was performed using hRFC (B) and hPCFT (E) gene-specific primers and total RNA (5 μg) from preconfluent, confluent, and postconfluent Caco-2 cells. Real-time PCR was performed as described in materials and methods. Data represent means ± SE of at least 3 independent sets of experiments and were normalized relative to β-actin and calculated using a relative relationship method supplied by the iCycler manufacturer (Bio-Rad, Hercules, CA). Note that the level of expression of hRFC and hPCFT in preconfluent cells was set at 1 for each figure and the expression during differentiation is in relation to that level; therefore the results are not representative of the levels of hRFC compared with hPCFT (hPCFT mRNA expression is ∼3-fold higher than hRFC at all 3 stages of differentiation). C and F: activity of full-length hRFC promoter B (C) and hPCFT promoters (F) in pGL3-Basic was determined following transient expression in preconfluent, confluent, and postconfluent Caco-2 cells. Luciferase activity was determined and normalized relative to the activity of simultaneously expressed Renilla luciferase as described in materials and methods. The results are expressed relative to the PGL3-Basic vector set at 1. Data represent means ± SE of at least 3 independent sets of determinations.

Carrier-mediated folic acid uptake by native mouse jejunum crypt and villus epithelial cells. Initial rate (10 min; data not shown) of carrier-mediated uptake of [³H]folic acid (9 nM) by crypt and villus intestinal epithelial cells isolated from jejunum of wild-type mice was examined in Krebs-Ringer buffer (pH 5.5). Data represent means ± SE of at least 3 independent uptake determinations performed on cells isolated from different mice.

Levels of expression of mouse RFC (mRFC) and mouse PCFT (mPCFT) protein and mRNA in native mouse jejunum crypt and villus epithelial cells. A and C: Western blot analysis was performed using membranous fractions (150 μg) separated from crypt and villus of mouse jejunum and hybridized with polyclonal antibodies specific for mRFC and mPCFT as described in materials and methods. Immunodetection was performed with an enhanced chemiluminescence detection system (Amersham). Images and data shown are representative of 3 separate sets of experiments on cells isolated from at least 4–6 mice. B and D: quantitative real-time PCR was performed using mRFC (B) and mPCFT (D) gene-specific primers and total RNA from crypt and villus wild-type mouse intestinal epithelial cells. Primers () and PCR conditions were used as previously described (, ). Data represent means ± SE of at least 3–8 separate determinations on cells isolated from 3–6 wild-type mice and were normalized relative to β-actin and calculated using a relative relationship method provided by a commercial vendor (Bio-Rad, Hercules, CA). Note that the level of expression of mRFC and mPCFT in crypt cells was set at 1 for each figure and the expression in villus is in relation to that level; therefore the results are not representative of the levels of mRFC compared with mPCFT (mPCFT mRNA expression is ∼3-fold higher than mRFC at both crypt and villus stages).

Immunofluorescence analysis of mRFC and mPCFT proteins in crypt and villus mouse jejunum sections. A and B: immunofluorescence analysis was performed using mouse jejunum sections (8 μm) and polyclonal antibodies specific for mRFC and mPCFT, respectively, as described in materials and methods (23). C: jejunum section was incubated only with secondary antibody. Data and images shown are representative of 3–4 separate sets of tissues from different mice.