FIGURE 7.

CBWD expression and response to increased extracellular zinc concentration. A, CBWD transcript levels in RNA from Caco-2 cells measured by quantitative RT-PCR and expressed relative to GAPDH. Confluent cells were maintained for 24 h, 24 h after seeding, in serum-free medium containing zinc sulfate added to 3 μm (control) or 100 μm, as indicated. Data are expressed as mean ± S.E. (error bars) for n = 6. ***, p < 0.001 by Student's unpaired t test; B, representative Western blot showing signal intensities for recombinant CBWD3 protein expressed in CHO cells with a C-terminal FLAG epitope tag and detected using anti-FLAG antibody and for α-tubulin (loading control) after maintenance of cells for 24 h in serum-free medium containing zinc sulfate added to 3 μm (control) or 100 μm, as indicated, 24 h after transfection with the CBWD3 expression construct (pCMV6Entry-CBWD3; Origene). Molecular weights are indicated. C, data derived by densitometric quantification of band intensities from Western blots as described for B, determined using UviPhotoMW image analysis software, expressed as the ratio of the CBWD1 signal to the α-tubulin signal and normalized to the control condition then expressed as mean ± S.E. for n = 7. *, p < 0.05 by Student's unpaired t test. D, effect of mutating the ZTRE motif on the response of the CBWD1 promoter to elevated extracellular zinc concentrations in Caco-2 cells. Caco-2 cells were transfected transiently with promoter-reporter plasmids containing the −1077 to +95 region of the CBWD1 promoter in which the ZTRE (positions −133 to −118) was either present (pBlueCBWDprom) or replaced with a random nucleotide sequence (pBlueCBWDprom_MUT). Twenty-four hours following transient transfection, Caco-2 cells were maintained in serum-free medium supplemented with either 3 μm (control) or 100 μm zinc, as indicated (added as ZnSO₄), for an additional 24 h. Promoter activity was measured as β-galactosidase reporter gene activity in cell lysates and expressed relative to protein concentration. Data are mean ± S.E. normalized to the control condition for n = 24 for pBlueCBWDprom and n = 12 for pBlueCBWDprom_MUT. ***, p < 0.001 by Student's unpaired t test. E, expression of CBWD in a panel of human tissues detected by RT-PCR. Primers specific to CBWD and to GAPDH (positive control) were used in PCRs as indicated after reverse transcription of total RNA samples from the tissues as identified. No products were observed when reverse transcriptase was omitted from reactions, confirming the origin of the products as mRNA (not DNA contamination). Product sizes are indicated.