1. Volume 114, Issue 6, Pages 1221-1229 (June 1998)
Differential activation of IGF-II promoters P3 and P4 in Caco-2 cells during growth and differentiation 
Pomila Singh, Bosong Dai, Randall L. Given, Xianbin Lu, P.Elly Holthuizen 
Gastroenterology 
Volume 114, Issue 6, Pages (June 1998)
DOI: /S (98)
Copyright © 1998 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Schematic representation of the human IGF-II gene and corresponding mRNAs. The numbered boxes indicate the 9 exons of the IGF-II gene (1–9). The four promoters P1–P4 and the two poly(A) addition signals (*) are indicated. Solid boxes represent exon sequences encoding preproIGF-II, and open boxes denote 5' and 3' untranslated sequences. Adapted and reprinted with permission.23
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Diagrammatic representation of the IGF-II promoter-luc constructs used in transient transfection assays. Specific restriction sites available in the P1–P4 promoters and exons 1, 2, and 4–6 were used for constructing the various luciferase expression vectors in pSla3 plasmid, as described in Materials and Methods. The promoter-luciferase constructs used are diagrammatically presented.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

4. Fig. 3
RPA with the exon 5 and 6 riboprobes. RNA samples from Caco-2 cells on different days of cell culture were hybridized with either the sense (S) or the antisense (AS) RNA probes for transcripts containing exon 5 (P3-derived transcripts) or exon 6 (P4-derived transcripts) sequences, as described in Materials and Methods. Ribonuclease-protected bands were separated by electrophoretic fractionation of the samples, and representative autoradiograms (of several similar autoradiograms from three experiments) are shown in A–C. (A) Samples hybridized with the 2× purified exon 5 probe. Lanes 1–5, day 2–5 and 7 RNA samples hybridized with AS exon 5 riboprobe; lane 6, day 4 RNA sample hybridized with sense exon 5 riboprobe; lane 7, exon 5 AS riboprobe; lane 8, exon 5 S riboprobe; lane 9, molecular weight markers. (B) RNA samples hybridized with exon 6 riboprobe. Lanes 1–6, day 2–7 samples hybridized with AS exon 6 riboprobe; lane 7, exon 6 AS riboprobe. (C) RNA samples hybridized with 28S riboprobe. Lane 1, 100-bp molecular weight marker; lanes 2–9, day 2–7 and 9, and 11 RNA samples hybridized with AS 28 S riboprobe; lane 10, AS 28S riboprobe.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Relative levels of IGF-II mRNA derived from P3 (■) and P4 (2) promoters on different days of cell culture. Densitometric readings of the 535-bp band (hybridized with the AS exon 5 probe, as shown in Figure 3A) and of the −105-bp band (hybridized with AS exon 6 riboprobe as shown in Figure 3B), were corrected for the corresponding ~115-bp band (hybridized with the 28S riboprobe, as shown in Figure 3C). Data thus obtained from three (days 2 and 9) or four (days 3–8) separate experiments are presented as percent of expression of day 3 values (wherein day 3 values were arbitrarily assigned a 100% value).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Transcriptional activity of HUP3 (■) and HUP4 (2) vectors on days 2–9 of cell culture. (A) Caco-2 cells on days 2–9 were cotransfected with the indicated promoter-luc expression vector DNA and the β-Gal vector DNA, and 24 hours later, luc and β-Gal activities were measured as described in Materials and Methods. Representative data from a single experiment (of three separate experiments) are presented as the ratio of luc/β-Gal activity and are the mean ± SEM of four separate dishes. (B) Total cell number in duplicate dishes (measured with a Coulter counter) are shown and represent an average of three measurements from three separate dishes per experiment. Cells in dishes on days 7 and 8 of cell culture seemed to be completely confluent (at the light microscope level); thus, 2.8 × 106 cells (measured on days 7 and 8 of cell culture/35-mm dish) were considered 100% confluent. The relative confluency of the dishes on days 2 and 7 was derived from the total cell number measured per dish on day of harvest, as shown. (C) Days 3–6 were assigned the phase of logarithmic cell growth, based on the fact that cells per dish doubled every 24 hours on these days. Days 7–10 were arbitrarily assigned the plateau phase of cell growth because during this time cells per dish increased by only 7%–20%/24 hours. Percentage of cells in the S phase was determined by immunocytochemical staining of cells positive for BrdU uptake as described in Materials and Methods. A rough estimate of the percent of cells that were positive for BrdU uptake during the three indicated phases of cell growth is shown. The corresponding ALP labeling is also shown. ALP labeling was determined to rapidly increase between days 7 and 10 of cell culture as shown. During the logarithmic growth phase, ALP staining was negligible, and this period of growth is presented as growth associated with negligible differentiation. On days 7 and 8, a significant increase in ALP staining was measured, and this phase of cell growth is presented as growth associated with an increase in differentiation. On days 9 and 10, labeling was further increased compared with that on days 7 and 8, and this phase of cell growth is presented as phase of rapid differentiation.
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Photomicrographs of Caco-2 cells labeled with ALPs and BrdU antibodies on different days of culture (original magnification, 220×). (A–C) Light micrographs of Caco-2 cells showing immunocytochemical labeling of incorporated BrdU. The labeling activity decreases with increasing time in culture. (D–F) Immunocytochemical labeling for the presence of alkaline phosphatase in Caco-2 cells. The brown reaction product is not visible on day 3 of culture, but becomes very apparent by day 10 (A and D, day 3; B and E, day 5; C and F, day 10).
Gastroenterology  , DOI: ( /S (98) )
Copyright © 1998 American Gastroenterological Association Terms and Conditions