1. PTF1α/p48 and cell proliferation
Annie Rodolosse, Elisabet Chalaux, Teresa Adell, Hélène Hagège, Anouchka Skoudy, Francisco X. Real 
Gastroenterology 
Volume 127, Issue 3, Pages (September 2004)
DOI: /j.gastro
Copyright © 2004 American Gastroenterological Association Terms and Conditions

2. Figure 1
Cell growth inhibition of pancreatic cancer cell lines by p48. Control (C) and p48 transfectants were seeded in 6-well culture plates at the same density. After 2 weeks, colonies were stained and counted as reported in the Materials and Methods section. Values are shown as mean ± SD of 3 independent experiments. For each cell line, 1 representative colony formation assay is shown. (A) AR42J cells. (B) RWP-1 cells seeded into soft agar.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

3. Figure 2
p48 Decreases the growth rate of pancreatic and nonpancreatic cells. (A) RWP-1 transfectants expressing, or not, p48 were seeded in 6-well plates. Cells were counted over a 7-day period. Each point corresponds to the mean ± SD of 4 values. (B) Stably transfected Cos-7 cells, obtained from 3 independent transfection experiments, were seeded in duplicate in 6-well plates and counted after 14 days, as reported in the Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

4. Figure 3
p48 does not affect cell death and causes a delay in G1-S progression. (A) 104 RWP-1 control (open bars) and p48 transfectants (solid bars) were seeded in 6-well plates and counted after trypan blue staining on culture day 3, 5, 6, and 7. Percentage of dead cells was determined by dividing the number of trypan blue-stained cells by total cell number. Results were obtained from 3 independent control and p48 transformant populations. (B) Western blotting analysis was performed with lysates from 2 RWP-1 control (C) or p48-transfected cell populations. Proteins of the expected size (32 kilodaltons and 17 kilodaltons) were detected with polyclonal anti-procaspase-3 and anti-cleaved caspase-3 antisera, respectively. Equal protein loading was confirmed by probing with an antibody against β-actin. (C and D) FACS analysis of cell cycle proliferation. RWP-1 transfectants expressing or not p48 were cultured in serum-deprived medium for 48 hours. Serum-deprived medium was then replaced by 10% serum containing medium. Cells were collected before (0% FBS, open bars) and 24 hours after medium change (10% FBS, solid bars) for PI staining and FACS analysis. Fractions of cells in G0/G1, S, G2/M phase were determined by FACS for each condition (0% FBS or 10% FBS) (C), and the proliferation index of control and p48 transfectants was calculated as reported in the Materials and Methods section (D). Data were obtained from 3 control and p48 transfectant populations in at least 4 independent experiments.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

5. Figure 4
Effects of p48 on the expression of cell growth regulators. Up-regulation of p21CIP1/WAF1 and p27KIP1. Western blotting analysis of G1 phase regulators was performed with whole-cell extracts (50 μg) prepared from stably transfected RWP-1 cells. Results shown were obtained with cell extracts of 2 control and 2 p48 transfectant populations obtained independently (designated 1 and 2, respectively).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

6. Figure 5
p21CIP1/WAF1 and cyclin D2 are regulated at the mRNA level. RT-PCR analysis was carried out with total RNA extracted from control of p48 RWP-1 transfectants. The amount of RNA used in RT-PCR reactions is indicated. The relative intensity of the bands corresponding to the RT-PCR products was quantitated by image analysis, and levels of p21CIP1/WAF1, p27KIP1, and cyclin D2 RT-PCR products were normalized against those of cyclophilin.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

7. Figure 6
p48 activates the p21CIP1/WAF1 promoter. (A) p48 activates the promoter of p21CIP1/WAF1 gene. pIRESneo control or pIRESp48 vector (0.4 μg) was cotransfected with 0.1 μg p21-luc reporter construct27 and 15 ng pRL-TK vector containing the herpes simplex virus thymidine kinase promoter upstream of Renilla luciferase cDNA in RWP-1 cells plated in 24-well plates. pRL-TK vector was included as an internal control for normalization. Luciferase expression was evaluated 48 hours after transfection. Relative luciferase activity values are the means of 2 independent experiments, each performed in triplicate. (B) p53 is not required for the antiproliferative activity of p48. RWP-1, 3T3 WT, and 3T3 p53−/− cells were transfected with pIRESneo (open bars) or pIRESp48 vector (solid bars) (0.4 μg). Twenty hours after transfection, cells were labeled with 1 μmol/L BrdU for 6–12 hours, and quantitation of BrdU incorporation was assessed by double immunostaining of BrdU and p48 as indicated in the Materials and Methods section.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

8. Figure 7
Structure and antiproliferative activity of deletion mutants of p48. (A) Structure of the p48 deletion mutants. (B) AR42J (open bars) and RWP-1 cells (solid bars) were stably transfected with expression vectors of the p48 deletion mutants represented in panel A. AR42J and RWP-1 proliferative capacity was determined by colony formation assays performed as described in Figure 1. The results shown were obtained from 2 independent experiments. (C) Transcriptional activation of the A element of the elastase promoter by p48 deletion mutants. AR42J cells were transiently cotransfected with the pIRESneo vector containing the full-length, or partially deleted, p48 and the 6XA26-luc reporter construct. pRL-TK vector was included as an internal control for normalization, and luciferase activity was determined 48 hours after transfection.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions

9. Figure 8
Cell proliferation in ductal complexes. (A) Comparison of the proportion of Ki-67 immunoreactive cells in paired samples of histologically normal pancreas (open bars) and regions containing ductal complexes typical of chronic pancreatitis (solid bars). Samples were obtained from 3 patients (designated 1, 2, and 3). The total number of cells counted ranged between 4000 and 20,000. Statistical analysis: *P = 0.02; **P < (χ2 test). (B—E) Cell proliferation and differentiation were assessed in paraffin-embedded pancreatic tissue sections using immunohistochemistry with antibodies recognizing Ki-67 or amylase. Areas corresponding to histologically normal pancreas (B) or an area containing ductal complexes (C) from a patient with chronic pancreatitis: less than 1% of proliferating cells are present in normal pancreas, whereas a higher proportion of proliferating cells is observed in chronic pancreatitis-associated ductal complexes. Transdifferentiated ductal complexes in pancreatic tissue from MT-TGF-α transgenic mice (D and E); Anti-Ki-67 (B—D); amylase (E).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2004 American Gastroenterological Association Terms and Conditions