Figure 4

In vitro virulence of Ehcp112-silenced trophozoites. (A) Protease activity. The protease activity of the trophozoites treated by 16 h with the siRNA 1 (EhCP112-silenced trophozoites) was analyzed in polyacrylamide-0.1% gelatin substrate gels. Lane 1, extracts from untreated trophozoites (Ctl+); lane 2, extracts from EhCP112-silenced trophozoites (SiRNA); lane 3, extracts from trophozoites treated with a non-related sequence (NRS). Arrow indicates the 62-kDa band with minor proteolytic activity in EhCP112-silenced trophozoites. Below is shown the densitometry of the proteinase activity of the 62- and 49-kDa bands. (B) Cytotoxic activity. Total extracts from EhCP112-silenced trophozoites (SiRNA) and NRS-treated trophozoites (NRS) were incubated with MDCK monolayers during 2 h. Then, the monolayer destruction was evaluated. (C) Cytopathic assays. EhCP112-silenced trophozoites (SiRNA) and NRS-treated trophozoites (NRS) were incubated with MDCK monolayers during 2 h. Then, the monolayer destruction was evaluated. (D) Erythrophagocytosis assays. EhCP112-silenced trophozoites (SiRNA) and NRS-treated trophozoites (NRS) were incubated with human red blood cells during 5 and 10 min. Then, the ingestion of the target cells was evaluated and value obtained with the NRS-treated trophozoites at 10 min of phagocytosis was arbitrary taken as 100% efficiency. Data were analyzed in three independent assays by duplicate and they are expressed as the means ± standard deviation. Differences were evaluated by t-student test. Asterisks: P < 0.001.