These genes come from different families, with different functions, so this shRNA knockdown method appears robust and not specific to only one gene or gene family. Methods Culture of trophozoites E. histolytica strain HM1:IMSS trophozoites were grown axenically in TYI-S-33 (Trypticase-yeast extract-iron-serum)

(TYI) medium supplemented with 1× Diamond’s vitamins (SAFC Biosciences, Lenexa, KS, USA), 15% Quizartinib manufacturer Heat-inactivated adult bovine GW786034 serum (Gemini Bio-Products, West Sacramento, CA), 100 U of penicillin/ml and 100 μg streptomycin sulfate/ml (Gibco/Invitrogen, Carlsbad, CA, USA), at 37°C in 25 cm2 tissue culture flasks [47] in a volume of 50 ml, and then transfected as described below. Transfection of amebae Plasmid DNA was prepared selleck chemical using the HiSpeed Qiagen Maxi Kit (Qiagen, Valencia, CA, USA). Medium 199 (M199) (Gibco BRL/Invitrogen, Carlsbad, CA, USA) was supplemented with 5.7 mM cysteine, 25 mM HEPES, and 0.6 mM ascorbic acid [48], adjusted to pH

7.0 and filter-sterilized. Twenty μg plasmid DNA diluted in 100 μl supplemented M199s medium (M199S) in 2-ml microcentrifuge tubes was mixed with 15 μl of SuperFect or Attractene transfection reagent (Qiagen, Valencia, CA, USA), and incubated at room temperature to allow transfection-complex formation as per the manufacturer’s instructions. Heat-inactivated bovine serum was added to the remaining M199S to a 15% concentration. Amebae were harvested by tapping the tissue culture flasks on a benchtop, were centrifuged at 200 × g for 5 min at 4°C, and suspended in M199S with serum to 2.5 × 105 amebae/ml. Tubes containing transfection complexes were filled with the suspended trophozoites, the contents mixed by inversion, and the tubes were incubated horizontally for 3 hours at 37°C. Tube contents were added to warm TYI in 25 cm2 tissue culture flasks, and incubated overnight at 37°C. 15 μg/ml hygromycin (Invitrogen, Carlsbad, CA, USA) was added for selection after the overnight incubation [49]. After 4–5 days, 25 ml of the TYI was removed to

a new 25 cm2 tissue culture flask, and 25 ml fresh TYI with hygromycin Plasmin was added to each of the flasks. Transfectants were usually apparent 1–2 weeks after transfection. E. histolytica shRNA constructs All short hairpin RNAs used in this study were expressed by the U6 promoter [GenBank:U43841] [41] (Figure 1A) and cloned into the amebic expression vector pGIR310, a modification of pGIR308 [49, 50] by the addition of a short polylinker containing HindIII, SalI, and NotI restriction sites (Figure 1B). Modified pGIR310 conferred resistance to hygromycin in E. histolytica and to ampicillin in Escherichia coli (E. coli). All shRNA constructs used in these studies had the same structure: a short hairpin consisting of a 29-nucleotide sense strand, followed by the 9-nucleotide loop and the 29-nucleotide complementary antisense strand (Figure 1).