Fig. 1. (A) Northern blot. Double-stranded RNA causes destruction of PEX2 mRNA. Each lane on the blot contains 10 µg poly(A)+ RNA from the procyclic form RNAi cell line. Cells were grown either without Tet (-), or for 13 hours in the presence of 100 ng/ml Tet (+). (B) Growth curves. Filled figures and unbroken lines, cells grown without Tet; open figures and dashed lines, cells grown in the presence of 100 ng/ml Tet. Diamonds, cells with pex2r downstream of the inducible RNA polymerase I promoter. The cultures were initiated at 1.5x10⁵ cells/ml and followed for 2 days. Squares, cells expressing the T7 polymerase and with a full-length PEX2 downstream of an inducible T7 promoter. Circles, the dominant-negative mutant — cells expressing the T7 polymerase and with the pex2r gene downstream of an inducible T7 promoter. Cultures of cells expressing the T7 polymerase were initiated at 5x10⁴ cells/ml. (C) Western blot. Digitonin treatment of the dominant-negative mutant. 10 µg of protein (1-2x10⁶ cells) were taken from each sample (cells grown with and without Tet) for treatment with 6 µg of digitonin. s, supernatant (cytosolic fraction); p, pellet (glycosomal fraction). The glycosomal proteins detected are indicated on the right. (D) Growth curves of RNA interference cells. Filled circles and unbroken lines, control — cells grown without Tet; open circles and broken lines, cells in the presence of 100 ng/ml Tet. Bloodstream cultures were initiated at 1x10⁵ cells/ml and daily diluted to the same concentration. Procyclic cultures were daily diluted to 5x10⁵ cells/ml.