<div><p>(A) HFF cells transfected with the siRNA SmartPool against CDA1 and co-treated with Compound 1 prior to parasite infection allow normal parasite growth (black boxes) and BAG1 expression (dark bars). Compare growth in untreated controls (black circles) with growth (grey boxes) and BAG1 expression (light bars) in Compound 1–treated cells.</p><p>(B) Concurrent with the restoration of normal parasite growth and BAG1 expression, we observed the loss of Compound 1–induced CDA1 mRNAs as measured by RT-PCR of total RNA at 24, 48 following transfection with siRNA and Compound 1 pretreatment. Lamin A/C controls demonstrate siRNA-mediated knockdown of individual genes. Transfection with siRNAs against Lamin A/C, with nonspecific siRNAs or with LipofectAMINE alone, was unable to antagonize Compound 1 inhibition of parasite growth or induction of BAG1 expression (results not shown). RT-PCR primers specific to GAPDH were used as a control for RNA quality. RT-PCR experiment: C = Compound 1 pretreatment (3 h/3 μM) only; E = HFF cells pretreated with both Compound 1 and siRNA(s) against either CDA1 or Lamin A/C. Protein blot: L = HFF cells pretreated with LipofectAMINE, but without siRNAs; si = HFF cells pretreated with siRNAs against either CDA1 or Lamin A/C, but without transfection reagent (LipofectAMINE); E = same as above.</p><p>(C) Co-transfection of HFF cells with siRNAs 1 to 4 separately and then treatment with Compound 1 prior to parasite infection demonstrate that siRNAs 2 and 3 were responsible for antagonizing the Compound 1–induced inhibition of parasite growth as measured by average parasites per vacuole at 48 h postinfection. Compare light bars for siRNAs 2 and 3 with untreated parasites (labeled no siRNA), and also with Compound 1–treated parasites (labeled Compound 1). siRNAs 1 and 4 do not significantly antagonize the Compound 1–induced inhibition of parasite replication.</p></div
