Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression

Additional File 1:

(A) E. histolytica HM-1:IMSS grown in 23 μM 5-AzaC for five consecutive days have similar growth kinetics to untreated parasites. E. histolytica HM-1:IMSS log-phase trophozoites (15,000) were seeded into 15 ml glass culture tubes, cultured under standard conditions ± 23 μM 5-AzaC, and cell counts taken at days 2, 3, 4, and 5. The average and standard deviation values for each time point are shown for untreated and 5-AzaC treated parasites. There were no statistical differences in the cell counts at any given time point in 5-AzaC treated and untreated parasites. One representative experiment is shown. (B) Protein content per cell was determined at days 2, 3, 4, and 5 of parasite growth ± 23 μM 5-AzaC treatment for E. histolytica HM-1:IMSS parasites. The average and standard deviation values for each time point are shown. There were no statistically significant differences in the protein concentration per cell at any given time point in 5-AzaC treated and untreated parasites or in parasites grown for 2, 3, 4, or 5 days. One representative experiment is shown. (C) E. histolytica HM-1:IMSS grown in 23 μM 5-AzaC for six days with routine subculturing of equal volume of media/parasites have similar growth kinetics to untreated parasites after two days in 5-AzaC. E. histolytica HM-1:IMSS log-phase trophozoites (50,000) were seeded into 15 ml glass culture tubes and cultured under standard conditions ± 23 μM 5-AzaC. At days 2 and 4 (marked with a down arrow) an equal volume of media containing parasites grown ± 5-AzaC was inoculated into a new tube with fresh media (± 5-AzaC) and cell counts obtained at days 2, 4, and 6. The average and standard deviation values for each time point are shown for untreated and 5-AzaC treated parasites. One representative experiment is shown.

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Normalized expression values for arrays from untreated and 5-AzaC treated E. histolytica HM-1:IMSS parasites. All array data have been made available per MIAME guidelines. Raw array data have been deposited at the NCBI Gene Expression Omnibus website (accession number GSE6635).

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Verification of array data for EHsp100 (represented on the microarray by the probe set 64.m00187_s_at) by semi-quantitative RT-PCR. Total RNA was isolated from untreated and 5-AzaC treated parasites (7 days) and subjected to RT-PCR. Sequential 1:100 dilutions of cDNA were used as template for the PCR and a genomic DNA and minus RT control (-RT) were included. The microarray expression fold-change for each gene is shown based on average array data from 3-day and 7-day 5-AzaC treated parasites. Primers specific to the EHsp100 gene (192.m00086) as determined by Bernes et al [16] were used for the RT-PCR reaction. A gene whose expression did not change based on array data (147.m00095) was found to be unchanged in the two conditions by RT-PCR.

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This Python script determines the frequencies of mono- and dinucleotides in a FASTA sequence file. This program requires a Python interpreter installed on the computer that is being used for this analysis, which can be freely downloaded [74]. Download the text of this file exactly as written (including white space). To run the program, type "python <name of script file> <name of FASTA sequence file>".

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