1. Antisense Inhibition of Entamoeba histolytica Cysteine Proteases Inhibits Colonic Mucus Degradation 
Darcym Moncada, Kathy Keller, Serge Ankri, David Mirelman, Kris Chadee 
Gastroenterology 
Volume 130, Issue 3, Pages (March 2006)
DOI: /j.gastro
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

2. Figure 1
Mucin degradation assays of wild-type and pSA8 transfected E histolytica, SDS-PAGE and autoradiograph of [35S]cysteine-labeled mucin from LS 174T cells. (A) Radiolabeled mucin (2 × 104 cpm/digest) was incubated with 100 μg of E histolytica cell lysate from wild-type (WT), pEhAct-neo grown in 12 μg/mL of G418 (Ct) or pSA8 transfectants grown in increasing concentrations of G418, for 6 hours at 37°C and separated by SDS-PAGE and visualized by autoradiography. (B) Digests were separated by gel filtration using a Sepharose 4B column. Mucin (1 × 105 cpm) in the absence of E histolytica lysate was used as a control. The column was calibrated with blue dextran (BD; 2000 kDa) and bovine serum albumin (BSA; 67 kDa, Amersham Biosciences, Uppsala, Sweden). (C) SDS-PAGE and autoradiograph of [35S]cysteine-labeled HT-29F Cl.16 mucin. Mucin alone as control (Ct), or treated with 50 μg of secreted products (SP) or SP and 100 μmol E-64. (D) S4B column chromatography of HT-29F Cl.16 mucin digests. Mucin in the absence of E histolytica secretory products was used as a control and the digests contained either secreted products as stated in (C) or pSA8-48 lysate as in (B).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

3. Figure 2
LS 174T cell monolayer destruction and mucin degradation by E histolytica cysteine proteases. (A) Destruction of an LS 174T cell monolayer by E histolytica trophozoites. Mucin-producing LS 174T cells were incubated with E histolytica trophozoites transfected with the pEhAct-neo (12 μg/mL G418) or pSA8 plasmids grown in 12, 24, and 48 μg/mL of G418. (B) The mucin-producing cell line, HT-29 Cl.16F was also used to assess monolayer destruction by the parasite after 3 hours of incubation. (C) CHO cell monolayers, which do not produce gel-forming mucin, were incubated for 3 hours with the transfectants as well as the pSA8-48 revertant (Rvt) grown in the absence of G418 for 3 months or in the presence of 100 μmol E-64 (E-64). For killing assays, the pSA8 transfectants were compared to the respective control (Ct) for each time point. *P < .05.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

4. Figure 3
Adherence of E histolytica transfectants to CHO cells. CHO cells were incubated with wild-type (WT) trophozoites (+) or with trophozoites preincubated with 1 μg of colonic mucin (−) for 1 hour. Adherence of pEhAct-neo (Ct), pSA8 transfectants, and pSA8-48 revertants (Rvt) was determined, as well as pSA8-48 and WT trophozoites in the presence of 50-mmol galactose. An E histolytica trophozoite adherent to 3 or more CHO cells was considered a positive rosette formation. *P < .05 compared with Ct.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

5. Figure 4
Degradation of colonic mucin by recombinant EhCP5. (A) Gelatin zymogram of activated recombinant EhCP5 in the absence of presence of E-64. Clear band indicates CP activity. (B) SDS-PAGE and autoradiograph of [35S]cysteine-labeled mucin digested with 50 μg of E histolytica–secreted products (SP), rEhCP5 (4 μg), and rEhCP5 preincubated with E-64 (100 μmol) for 6 hours. The percent degradation was calculated as compared to the control mucin alone by densitometric analysis (Ct). (C) Sepharose 4B chromatography of [35S]cysteine-labeled mucin under the same conditions as (B).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

6. Figure 5
Secretion of [3H]-labeled mucin from colonic cells in response to E histolytica–secreted products. LS 174T (A) and HT-29F Cl.16 (B) colonic cells were incubated with 20 mmol calcium ionophore A (+) as a positive control for mucus secretion, or various concentrations of secreted products, and secreted products with 100 μmol E-64. Mucus secretion was assessed after 4 hours incubation at 37°C. (C) A coculture system of LS 174T cells with wild-type (WT) trophozoites and transfectants was used to measure mucus secretion in response to live parasites. Mucus secretagogue activity was plotted as the percent secretion of control. No significant differences in secretion are evident between the trophozoites. (NS) no significant difference between the two samples (±E-64). *P < .05 compared to baseline secretion (media alone).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions