Eukaryotic translation initiation factor 5A and its posttranslational modifications play an important role in proliferation and potentially in differentiation of the human enteric protozoan parasite Entamoeba histolytica
Fig 5
Effects of EhDHS2 gene silencing on gene expression of related genes and growth of E. histolytica trophozoites.
(A) Evaluation of gene expression by semi-quantitative RT-PCR analysis of EhDHS2 gene silenced transformant. The steady-state levels of transcripts of EhDHS1, EhDHS2, EheIF5A1, EheI5FA2 and EhRNA pol II genes were measured in trophozoites of G3 strain transfected with either empty vector (psAP2G) or the EhDHS2 gene silencing plasmid (psAP2G-DHS2). cDNA from the generated cell lines (psAP2G and DHS2gs strains) was subjected to 30 cycles of PCR using specific primers for the DHS2, DHS1, eIF5A1, eI5FA2 and RNA pol II genes. RNA polymerase II served as a control. PCR analysis of samples without reverse transcription was used to exclude the possibility of genomic DNA contamination. The densitometric quantification of the bands, shown in the right graph, was performed by Image J software, and the expression level of EhDHS1, EhDHS2, EheIF5A1, EheIF5A2, and EhRNA pol II was expressed in arbitrary units. (B) Growth kinetics of control (pSAP2G) and EhDHS2 gene silenced (DHS2gs) transformants. Approximately 60,000 amoebae in the logarithmic growth phase were inoculated into 6 mL fresh culture medium and amoebae were then counted every 24 hr. Data shown are the means ± standard deviations of five biological replicates. (C) Immunoblot analysis of control (pSAP2G) and EhDHS2 gene silenced (DHS2gs) transformants using hypusine antibody. Total cell lysate was electrophoresed on a 15% SDS-PAGE gel and subjected to an immunoblot assay with hypusine antibody and CS1 (loading control) antiserum. The intensity of the bands corresponding to hypusinated EheIF5A and CS1 was measured by densitometric quantification, analyzed by Image J software, and is shown in arbitrary units in the right graph.