1. Volume 123, Issue 1, Pages 196-205 (July 2002)
RhoA inactivation inhibits cell migration but does not mediate the effects of polyamine depletion 
Ramesh M. Ray, Anami Patel, Mary Jane Viar, Shirley A. Mccormack, Yi Zheng, Gabor Tigyi, Leonard R. Johnson 
Gastroenterology 
Volume 123, Issue 1, Pages (July 2002)
DOI: /gast
Copyright © 2002 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Polyamine depletion inhibits cell migration and decreases the level of RhoA protein. A confluent monolayer of cells was scratched with a razor blade and the migration assay was performed as described in the Materials and Methods section. (A) Migration was expressed as the number of cells crossing the scratch line per mm. Values are means ± SE of 6 observations. *Significantly different from control (P < 0.05). (B) Levels of active Rho (GTP-Rho), total RhoA, Rho-GAP, Rho-GDI, and -actin protein. A representative Western blot from 6 observations is shown. (C) Levels of RhoA protein (■) and RhoA activity (□) of the densitometric analysis of Western blots from an earlier experiment (B) is shown. Values are means ± SE of 6 observations. *Significantly different from control (P < 0.05).
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Polyamine depletion decreases RhoA protein levels in both cytosol and membrane fractions. Equal amounts of proteins were resolved by SDS-PAGE and subjected to Western blot analysis for the detection of RhoA protein. A representative Western blot and densitometry readings from 6 observations are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Effect of polyamine depletion on RhoA mRNA levels. A total of 30 μg RNA from each sample was resolved by agarose gel electrophoresis and subjected to Northern blot analysis for the detection of RhoA mRNA level. A representative Northern blot from 6 observations is shown.
Gastroenterology  , DOI: ( /gast )
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5. Fig. 4
Polyamine depletion does not affect the levels of Rac protein and mRNA. (A) Levels of Rac1 protein in the cytosol and membrane fraction. (B) A total of 30 μg RNA was subjected to Northern blot analysis by using Rac cDNA as a probe. Representative Northern and Western blots from 6 observations are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Polyamine depletion does not affect the stability of RhoA protein. Cell lysates containing equal amounts of radioactivity were used to immunoprecipitate RhoA. A representative gel from 3 observations is shown. Values are mean ± SE of 6 observations.
Gastroenterology  , DOI: ( /gast )
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7. Fig. 6
Polyamine depletion decreases RhoA translation. Cell lysates equalized for the amount of protein were used to immunoprecipitate RhoA. A representative gel from 3 observations is shown. Values are mean ± SE of 6 observations. *Significantly different from control (P < 0.05).
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions

8. Fig. 7
Recombinant RhoA protein expression influences IEC-6 cell migration. Confluent monolayers were wounded with a gel-loading tip in the center of plates marked to localize the wound site. Plates were photographed immediately to record the wound size (0 hour). At timed intervals, plates were photographed at the marked wound location. A representative of 3 experiments is shown.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Activated recombinant RhoA does not restore cell migration in polyamine-depleted cells. HA-V14–RhoA–transfected cells were grown in the presence and absence of 5 mmol/L DFMO and serum starved for 24 hours. Wounds are shown at 0 and 4 hours for control and DFMO-treated cells. Panels are representative of 3 experiments.
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
Polyamine depletion does not alter RhoA activation in HA-V14–RhoA or HA-N19–RhoA cells but inhibits RhoA activation in empty vector (Vector)-transfected IEC-6 cells. Activated RhoA protein bound to target peptide was separated by SDS-PAGE. Western blot analysis was performed by using RhoA antibody to determine the level of activated RhoA protein in the cell extracts. Values are mean ± SE of 6 observations. *Significantly different from control (P < 0.05).
Gastroenterology  , DOI: ( /gast )
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11. Fig. 10
Polyamine depletion alters the F-actin cytoskeleton of IEC-6 cells transfected with empty vector and RhoA. Vector, HA-V14–RhoA, and HA-N19–RhoA cells were grown under conditions for migration assays as described in Figure 8. Confluent cultures were wounded with a gel-loading tip and 8 hours later were fixed and stained with Texas red phalloidin for the localization of F-actin. Representatives of 3 experiments are shown.
Gastroenterology  , DOI: ( /gast )
Copyright © 2002 American Gastroenterological Association Terms and Conditions