Results: 4

1.
Figure 2

Figure 2. RT-PCR analysis of recombinant CFTR expression in the complemented CFBE41o− clones. From: Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels.

A. Expression of wtCFTR was prominent in the two stable cell clones c7-6.2wt and c10-6.2wt. B. Using allele-specific primer to detect the expression of the recombinant ΔF508 construct showed prominent expression in clone c4-4.7ΔF, but not in the other clones. C&D. Positive and negative controls are the expression of β-actin and processing the sample without reverse transcriptase (−RT), respectively. The primer pair for wtCFTR amplification was CF7C/CF17; expression of recombinant ΔF508CFTR was detected by primer pair CF81C2/CF17 specific for the TTT deletion in the construct (see Table 1 for sequences).

Beate Illek, et al. Cell Physiol Biochem. ;22(1-4):57-68.
2.
Figure 1

Figure 1. Immunohistochemical analysis of parental and complemented CFBE41o− cells. From: Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels.

Parental CFBE41o− cells and three clonal isolates expressing either the ΔF508CFTR construct (clone c4-4.7ΔF) or the 6.2kb wtCFTR construct (clones c7-6.2wt and c10-6.2wt). Cells were stained with FITC-tagged primary antibodies against ZO-1, K-18, and the SV40 large T antigen. A. Localization of ZO-1 to the plasma membrane at points of cell-cell contacts is consistent with the formation of tight junctions and maintenance of cell polarity. B. Staining for K-18 indicates a well-organized keratin filament structure in all cell lines. C. All cell clones stained positive for the SV40 large T antigen; original magnification 600×.

Beate Illek, et al. Cell Physiol Biochem. ;22(1-4):57-68.
3.
Figure 4

Figure 4. Expression of ΔF508CFTR mRNA and cAMP-dependent Cl transport in ΔF508CFTR complemented CFBE41o− cells. From: Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels.

A. The mean number of ΔF508CFTR plasmids per cell in CFBE41o− c4-4.7ΔF. Copy number was determined over 5 consecutive passages. The absolute passage numbers were p4.72.44 to p4.72.49. Subculturing did not affect the number of vectors per cell (p=0.56). B. The expression of ΔF508CFTR mRNA in clone c4-4.7ΔF relative to the level of wtCFTR expression in 16HBE14o− cells over 8 consecutive passages. The absolute cell culture passage numbers for c4-4.7ΔF were p4.72.41 to p4.72.48. There was no significant trend between CFTR mRNA expression and passage number (p=0.9). C. No significant forskolin-stimulated or GlyH101-sensitive chloride currents (ICl) were detected in the parental CFBE41o−. D. The c4-4.7ΔF clone consistently expressed small CFTR-mediated currents. E. A A summary of forskolin-stimulated and GlyH101-blocked Cl currents (ΔICl) in parental CFBE41o− (open bars) and c4-4.7ΔF (filled bars). Small but significant CFTR-mediated currents were induced by expression of recombinant ΔF508CFTR, p<0.001 for forskolin-activated currents, p=0.043 for GlyH101-blocked currents, n=7. The responses to these compounds in the parental CFBE41o− were not significantly different from 0 (n=7, one-sample t tests).

Beate Illek, et al. Cell Physiol Biochem. ;22(1-4):57-68.
4.
Figure 3

Figure 3. Quantitative assessment of CFTR mRNA expression and cAMP-dependent Cl transport in wtCFTR complemented CFBE41o− clones. From: Cl Transport in Complemented CF Bronchial Epithelial Cells Correlates with CFTR mRNA Expression Levels.

A. The mean number of plasmids per cell (n=3 per bar). Copy number was determined from cells that were 5 passages apart, passage numbers are given next to each symbol. The absolute number of passages (P) as denoted by “(passages after primary isolation).(passages after immortalization).(passages after CFTR transfection)” were p4.77.47 to p4.77.52 for c10-6.2wt, and p4.77.8 to p4.77.13 for c7-6.2wt. Subculturing did not affect the number of vectors per cell; c10-6.2wt expressed significantly higher levels of vector per cell than c7-6.2wt (ANOVA, p=0.005). B. CFTR expression by real-time PCR relative to that measured in the 16HBE14o− cells, relative passage numbers are as indicated. There was no change of expression with passage number (as determined by QRT-PCR analysis of expression level over passage number; c10-6.2wt, p=0.55; c7-6.2wt p=0.68); c10-6.2wt expressed significantly higher levels of mRNA compared to c7-6.2wt (p<0.001, paired t tests). The number of subcultures (P) was p4.77.4 to p4.77.11 for c10-6.2wt and p4.77.5 to p4.77.12 for c7-6.2wt. C. Vector number and CFTR mRNA levels correlated closely (0.9±0.1 mRNA increase per vector). D–F. Transepithelial recordings in the presence of a serosal-to-mucosal Cl gradient. Chloride currents (ICl) were activated by forskolin (20 µM) and inhibited by GlyH101 (20 µM) in c10-6.2wt (D), c7-6.2wt (E), and, for comparison, in 16HBE14o− (F). G. A summary of forskolin-stimulated and GlyH101-blocked chloride currents (ΔICl), n=4–10 experiments per bar; * denotes significant difference (ANOVA). H. The relationship between CFTR expression and function. The correlation between the GlyH101-blocked current and the relative CFTR mRNA expression resulted in a slope of 3.8±0.7 µA/cm2 per unit increase in CFTR mRNA levels, where one unit corresponds to the level of CFTR mRNA in 16HBE14o− cells.

Beate Illek, et al. Cell Physiol Biochem. ;22(1-4):57-68.

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