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. 2017 Aug 16;7:372. doi: 10.3389/fcimb.2017.00372

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Copyright © 2017 Cuellar, Hernández-Nava, García-Rivera, Chávez-Munguía, Schnoor, Betanzos and Orozco.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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EhCP112 overexpression increases TEER dropping without affecting cell viability. (A) RT-PCR analysis of stably-transfected trophozoites with (lane 1) pNeo or (lane 2) pNeoEhcp112 construction; (lane 3) pNeoEhcp112 plasmid; and (lane 4) PCR mixture without DNA, showing the neo and s2 control transcripts. (B) pNeoEhcp112 and pNeo transfected trophozoites were lysed and submitted to WB and revealed using the antibodies indicated at left. (C) Densitometry analysis of the data shown in (B), using actin as control. (D) Immunofluorescence assays of pNeoEhcp112 or pNeo transfected trophozoites incubated with α-EhCP112 and secondary-FITC antibodies. Nuclei were stained with PI. Bar = 10 μm. (E) Caco-2 cells were incubated with (□) pNeo or () pNeoEhcp112 or (■) pNeoEhcp112 transfected trophozoites pre-incubated with α-EhCP112 antibody and TEER was measured at 30 and 60 min. TEER values were normalized according to the initial value given by each transwell (1,000 Ω/cm²). Means and standard errors are represented for each time point of an assay performed by triplicate. ^*p ≤ 0.01; ^***p ≤ 0.001. (F) Caco-2 cells viability was measured by Sytox reagent. Upper panels show images of the Sytox treated cell monolayers.

Inline graphic — EhCP112 overexpression increases TEER dropping without affecting cell viability. (A) RT-PCR analysis of stably-transfected trophozoites with (lane 1) pNeo or (lane 2) pNeoEhcp112 construction; (lane 3) pNeoEhcp112 plasmid; and (lane 4) PCR mixture without DNA, showing the neo and s2 control transcripts. (B) pNeoEhcp112 and pNeo transfected trophozoites were lysed and submitted to WB and revealed using the antibodies indicated at left. (C) Densitometry analysis of the data shown in (B), using actin as control. (D) Immunofluorescence assays of pNeoEhcp112 or pNeo transfected trophozoites incubated with α-EhCP112 and secondary-FITC antibodies. Nuclei were stained with PI. Bar = 10 μm. (E) Caco-2 cells were incubated with (□) pNeo or () pNeoEhcp112 or (■) pNeoEhcp112 transfected trophozoites pre-incubated with α-EhCP112 antibody and TEER was measured at 30 and 60 min. TEER values were normalized according to the initial value given by each transwell (1,000 Ω/cm²). Means and standard errors are represented for each time point of an assay performed by triplicate. ^*p ≤ 0.01; ^***p ≤ 0.001. (F) Caco-2 cells viability was measured by Sytox reagent. Upper panels show images of the Sytox treated cell monolayers.