Fig 1.
The involvement of EGFR activation in Giardia-induced IEC apoptosis.
Unless otherwise specified, HT-29 and Caco-2 cells were exposed to Giardia trophozoites for the indicated time periods. (A) Giardia exposure promoted activation of PARP and CASP3 in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B, C) Apoptotic effects on HT-29 and Caco-2 cells by Giardia exposure were assessed by AO/EB staining (scale bar = 1000 μm) and Annexin V-FITC/PI staining. (D, E) Giardia exposure promoted activation of EGFR in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (F) PARP and CASP3 activation in HT-29 cells induced by a 6-h Giardia exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by western blot and gray value analyses. The comparisons were made as indicated. (G, H) HT-29 cell apoptosis induced by a 6-h Giardia exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by AO/EB staining (scale bar = 1000 μm) and Annexin V-FITC/PI staining. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01. “Ctr” read “Control”, “GI” read “Giardia”.
Fig 2.
EGF-like domains in Giardia tenascins and the interaction of tenascins with EGFR.
(A) Schematic diagram exhibiting the EGF-like domains of Tenascin15/30/33 (GenBank accession no. GL50803_114815/10330/16833) derived from G. duodenalis WB isolate (assemblage A) and the alignment of them with a human-derived biologically active EGF-like domain (GenBank accession no. KI723_040922) by ClustalW included in BioEdit v7.0.9 using default parameters [65] and then manual correction. (B, C, D) Effects of rTenascin15/30/33 on cell viability of HT-29 by CCK-8 assay.
Fig 3.
Ultrastructural analysis of Giardia/rTenascin15/30/33-unexposed and -exposed IECs by TEM.
HT-29 and Caco-2 cells were unexposed or exposed to Giardia trophozoites at a multiplicity of infection of 10 and rTenascin15/30/33 at a concentration of 20 μg/mL for 6 h. The exposed cells were examined for early and late apoptotic changes in contrast to unexposed controls (scale bar = 2 μm). “Control” was abbreviated as “Ctr”, “Nucleus” as “Nu”, and “Giardia” as “GI” here.
Fig 4.
rTenascin15/30/33 exposure induced IEC apoptosis.
rTenascin15/30/33 at a concentration of 20 μg/mL was applied. (A, D, G) Exposure of HT-29 and Caco-2 cells to rTenascin15/30/33 promoted activation of PARP and CASP3 as assessed by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B, C, E, F, H, I) Apoptotic effects on HT-29 and Caco-2 cells by rTenascin15/30/33 exposure were assessed by AO/EB (scale bar = 1000 μm) staining and Annexin V-FITC/PI staining. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.
Fig 5.
The involvement of rTenascin15/30/33 in EGFR activation.
Exposure of HT-29 and Caco-2 cells to rTenascin15/30/33 at a concentration of 20 μg/mL was able to promote activation of EGFR as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01.
Fig 6.
The association of EGFR activation with rTenascin15/30-induced IEC apoptosis.
rTenascin15/30/33 was used for exposure at a concentration of 20 μg/mL. (A, D) PARP and CASP3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478 as assessed by western blot and gray value analyses. The comparisons were made as indicated. (B, C, E, F) HT-29 cell apoptosis induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478 as determined by AO/EB (scale bar = 1000 μm) and Annexin V-FITC/PI analyses. (G) Activation of PARP and CASP3 in HT-29 cells induced by a 3-h rTenascin33 exposure could not be blocked by application of the EGFR inhibitor Ag1478 as determined by western blot and gray value analyses. (H, I) HT-29 apoptosis induced by a 3-h rTenascin33 exposure could not be blocked by application of the EGFR inhibitor Ag1478 as determined by AO/EB (scale bar = 1000 μm) and Annexin V-FITC/PI analyses. Data from triplicate wells from a representative of three independent experiments are presented as means ± SD. ** p < 0.01. “Control” was abbreviated as “Ctr”, and “not significant” as “ns” here.
Fig 7.
rTenascin15/30 exposure promoted EGFR translocation to the nucleus of IECs.
HT-29 cells were exposed to rTenascin15/30/33 at a concentration of 20 μg/mL. (A, B, C) Colocalization of rTenascin15/30/33 with EGFR was analyzed by immunofluorescent staining (scale bar = 50 μm). (D, E, F) rTenascin15/30 exposure promoted EGFR translocation to the nucleus, while this is not the case for rTenascin33, as determined by immunofluorescent staining (scale bar = 50 μm).
Fig 8.
Regulation of EGFR/STAT3 axis in rTenascin15/30-induced IEC apoptosis.
HT-29 and Caco-2 cells were exposed to rTenascin15/30/33 at a concentration of 20 μg/mL. (A) recombinant tenascin exposure promoted STAT3 activation in HT-29 and Caco-2 cells as determined by western blot and gray value analyses. The comparisons were made with the first group of the graph. (B) STAT3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the EGFR inhibitor Ag1478, while this is not the case for that induced by a 3-h rTenascin33 exposure, as assessed by western blot and gray value analyses. The comparisons were made as indicated. (C) PARP and CASP3 activation in HT-29 cells induced by a 6-h rTenascin15/30 exposure could be reversed by application of the STAT3 inhibitor Stattic, while this is not the case for that induced by a 3-h rTenascin33 exposure, as assessed by western blot and gray value analyses. The comparisons were made as indicated. (D) rTenascin15/30 exposure promoted EGFR/STAT3 translocation to the nucleus of HT-29 cells, while this is not the case for rTenascin33, as assessed by western blot and gray value analyses. The comparisons were made with the first group of the graph. Data from triplicate wells from a representative of at least three independent experiments are presented as means ± SD. * p < 0.05, ** p < 0.01. “Ctr” read “Control”, “ns” read “not significant”.