1. The homeodomain protein Cdx2 regulates lactase gene promoter activity during enterocyte differentiation 
Rixun Fang, Nilda A. Santiago, Lynne C. Olds, Eric Sibley 
Gastroenterology 
Volume 118, Issue 1, Pages (January 2000)
DOI: /S (00)
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2. Fig. 1
Sequence of wild-type and mutated CE-LPH1 cis element region of the rat lactase gene promoter. The sequence of the wild-type rat CE-LPH1 cis element region is shown on the top. Double-stranded mutations corresponding to bold bases were incorporated into synthetic oligonucleotides for gel shift analysis (see Figure 3; Mut-1, -2, -3, and -4) and into site-directed mutagenesis reporter constructs (see bFigures 5 and 6; pM1Luc, pM2Luc, pM3Luc, and pM4Luc). The caudal-related protein consensus binding site, TTTA(C/T), is underlined.
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3. Fig. 2
Relative luciferase activity of intestinal and nonintestinal cells transfected with lactase 5' genomic deletions cloned upstream of a reporter gene. Subconfluent Caco-2 (▩) and L (2) cells were transfected with promoterless pGL3 and with lactase gene/luciferase reporter constructs (p3kLuc, p2kLuc, p800Luc, and p200Luc), which contain the −3 kb, −2 kb, −0.8 kb, and −0.2 kb fragments of the lactase 5'-flanking region, respectively, cloned upstream of a firefly luciferase reporter gene on pGL3 (schematics). Transfection efficiencies were normalized to renilla luciferase expression of a cotransfected pRL-CMV vector and expressed as relative luciferase activity (means ± SD; n = 3).
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4. Fig. 3
Electrophoretic mobility shift assay with Caco-2 cell nuclear extract and CE-LPH1 radiolabeled probe. Caco-2 cell nuclear extract protein (10 μg) was incubated with 32P-labeled CE-LPH1 double-stranded oligonucleotide probe alone (−) or in the presence of 100× unlabeled specific wild-type (LPH1), nonspecific (Sp1), or mutant (Mut1, Mut2, Mut3, and Mut4; sequences shown in Figure 1) oligonucleotides. Samples were loaded on a 6% acrylamide gel. NS, nonspecific.
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5. Fig. 4
Gel supershift with CE-LPH1 probe and anti-Cdx2 or nonimmune serum. Gel retardation was carried out with nuclear extract (10 μg) from Caco-2 cells and 32P-radiolabeled CE-LPH1 probe. The reaction mixture was incubated for 30 minutes at room temperature alone (− lane) or with 1.0 μL of mCdx2 antibody or nonimmune serum.
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6. Fig. 5
Cdx2 dose-dependent activation of lactase minimal promoter construct. Subconfluent Caco-2 cells were cotransfected with p200Luc and the molar mounts indicated of either the empty expression vector (pAlpha+) or the expression construct in which the rat Cdx2 cDNA was cloned in either the sense (prCdx2) or antisense orientation (prCdx2-Rev). Transfection efficiencies were normalized to renilla luciferase expression of a cotransfected pRL-CMV vector and expressed as relative luciferase activity (means ± SD; n = 3).
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7. Fig. 6
Cdx2 activation of wild-type and mutant CE-LPH1 reporter constructs in intestinal cells. Subconfluent Caco-2 cells were transfected with 0.6 pmol wild-type (p200Luc) or mutant lactase gene/luciferase reporter constructs (pM1Luc, pM2Luc, pM3Luc, and pM4Luc) in the presence or absence of 0.3 pmol of the rat Cdx2 expression plasmid, prCdx2. Cells transfected in the absence of prCdx2 were instead cotransfected with pAlpha+ as a control. Relative luciferase activity of intestinal Caco-2 cells transfected with wild-type and mutant CE-LPH1 reporter gene constructs (▩) and cotransfected with Cdx2 expression construct (■) was assayed 48 hours after transfection. Transfection efficiencies were normalized to renilla luciferase expression of a cotransfected pRL-CMV vector and expressed as relative luciferase activity (means ± SD; n = 3).
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8. Fig. 7
Cdx2 activation of wild-type and mutant CE-LPH1 reporter constructs in nonintestinal cells. Subconfluent L cells were transfected with 0.6 pmol wild-type (p200Luc) or mutant lactase gene/luciferase reporter constructs (pM1Luc, pM2Luc, pM3Luc, and pM4Luc) in the presence (■) or absence (▩) of 0.3 pmol of the Cdx2 expression plasmid prCdx2. Transfection efficiencies were normalized to renilla luciferase expression of a cotransfected pRL-CMV vector and expressed as relative luciferase activity (means ± SD; n = 3).
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9. Fig. 8
Lactase and Cdx2 mRNA abundance and lactase enzymatic during Caco-2 cell differentiation. Total RNA (1.0 μg) extracted from Caco-2 cells 2 days before confluency (day −2), at confluency (day 0), and after confluency (days 7, 14, and 28) was reverse transcribed and PCR amplified with either lactase- or GADPH-specific primers. A single PCR-amplified product of the exact predicted size was visualized for both primer sets after ethidium bromide staining of a 2% agarose gel. The negative gel images are shown on the top 2 panels. Total RNA (10 μg) from the same time points was analyzed by Northern blot hybridization with a 32P-labeled 600-bp rat Cdx2 cDNA probe. A single 2.0-kb band was visualized after autoradiography for 24 hours at −80°C (Cdx2 panel).
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10. Fig. 9
Lactase promoter-reporter construct activity in pre- and postconfluent transfected Caco-2 cells. Stable cell lines harboring the 2-kb, 800-bp, and 200-bp lactase-luciferase constructs or the promoterless pGL3 vector were harvested 2 days before confluency (▩) and 14 days after confluency (■) and assayed for luciferase activity relative to cell lysate total protein level (means ± SD; n = 3).
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11. Fig. 10
Electrophoretic mobility shift assay with nuclear protein from different stages of enterocyte differentiation. Nuclear extract protein was isolated from Caco-2 cells just reaching confluency (day 0), 1 week after confluency (day 7), and 2 weeks after confluency (day 14). Caco-2 cell nuclear extract protein (5 μg) was incubated with 32P-labeled CE-LPH1 double-stranded oligonucleotide probe alone (−) or in the presence of 100× unlabeled specific CE-LPH1 (+). Samples were loaded on a 6% acrylamide gel. NS, nonspecific.
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12. Fig. 11
Gel supershift with Cdx2 antibody and CE-LPH probe during Caco-2 differentiation. Gel retardation was carried out with nuclear protein (5 μg) extracted from Caco-2 cells 2 days before confluency (day −2), at confluency (day 0), and after confluency (days 7, 14, and 28) and the 32P-radiolabeled CE-LPH1 probe. The reaction mixture was incubated for 30 minutes at room temperature alone (− lane) or with 1.0 μL of rat Cdx2 antibody (Cdx2 lane) or preimmune serum (Pre lane) or in the presence of 25× excess unlabeled specific wild-type CE-LPH1 (day 28, X lane). Eight-hour exposure of the day 14 gel retardation is shown at right (Day 14* lanes).
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13. Fig. 12
Lactase mRNA abundance in Caco-2 cells overexpressing Cdx2. Total RNA (1.0 μg) extracted from preconfluent Caco-2 cells transiently transfected with the prCdx2 expression construct or with the negative control pAlpha+ vector was reverse transcribed and PCR amplified with either lactase- or GADPH-specific primers. A single PCR-amplified product of the exact predicted size was visualized for both primer sets after ethidium bromide staining of a 2% agarose gel and is shown as the negative image in the upper 2 panels. Total RNA (10 μg) from the same transfected cells was analyzed by Northern blot hybridization with the 32P-labeled 600-bp rat Cdx2 cDNA probe. A single 2.0-kb band was visualized after autoradiography for 24 hours at −80°C (Cdx2 panel).
Gastroenterology  , DOI: ( /S (00) )
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