Figure 1.
Time-course of DNA-fragment transport across CaCo-2 cells.
CaCo-2 cells on filters were incubated with a 633 bp long polymerase chain reaction (PCR) amplified fragment and Lucifer yellow (LY) at 37 °C and samples were collected at the time points indicated. A: The amount of DNA-fragments transported across the cells in the apical to basolateral (AB) direction and the B
A direction was quantified by real-time PCR (qPCR) and normalized to the amount of DNA initially added to the cells and plotted against time. B: The amount of DNA-fragment left in the donor chambers after 90 min of incubation was quantified by qPCR and normalized to the amount of DNA initially added. C: All liquid in the basolateral donor chamber was collected from two wells and pooled before purification of DNA. PCR using the primers RRS SphI F and RRS SphI R was performed on the purified DNA before visualization on a 2% agarose gel to detect the full length DNA-fragment. D: After 90 min of incubation the amount of transcytosed LY was normalized to the amount of initially added LY in wells with or without addition of DNA-fragment. In A, B and D, the data shown are from one representative experiment with three replicates, showing mean +/−SD.
Figure 2.
Differentiation of CaCo-2 cells.
A: Trans-epithelial electric resistance (TEER) was measured on CaCo-2 cells on filters during their differentiation. Measurements were performed before change of medium. TEER (Ω x cm2) was plotted against time. One representative experiment is shown with mean +/−SD from nine wells. B: Intestinal alkaline phosphatase (IAP) expression at mRNA level detected by reverse transcription followed by PCR in CaCo-2 cells, CaCo-2/HT29-MTX Mix (3∶1) and HT29-MTX cells. Control is HeLa total mRNA from the Superscript III cellsdirect cDNA synthesis kit (Invitrogen). One representative experiment out of two is shown.
Table 1.
Effect of inhibition of endocytosis on transcytosis of DNA-fragment (5 nM).
Table 2.
Competition for transcytosis of DNA-fragment (5 nM) by nucleic acids and anionic compounds.
Table 3.
Transcytosis of Lucifer yellow (LY) in the presence of compounds affecting transcytosis of DNA-fragment.
Table 4.
Trans-epithelial electric resistance (TEER) in the presence of compounds increasing Lucifer yellow (LY)-transport.