A shRNA directed against green fluorescent protein (GFP) [30], with a sequence matching nothing in the E. histolytica genome, was utilized as a selleck chemicals control for transfection and hygromycin selection for the Igl and URE3-BP transfectants. GFP shRNA transfectants were selected with the same level of hygromycin as other shRNA transfectants. For EhC2A, a scrambled control matching nothing in the E. histolytica genome was created, containing the same nucleotides as the EhC2A (363–391) shRNA,

but in a different order. Sequences of the shRNA sense strands are shown in Table 1. Non-transfected HM1-IMSS amebae were also included, with the results for Western blotting and qRT-PCR being statistically the same as the GFP controls. Three biological replicates were grown per shRNA transfectant, and one for the nontransfected HM1:IMSS amebae. All sample trophozoites were grown in 25 cm2 tissue culture flasks, and were harvested for crude lysate buy BKM120 and for RNA isolation on the same day from the same flask. For protein and mRNA comparison, actin was used as the “”housekeeping”" control gene, as a loading and normalization control. Knockdown of Igl protein Four Igl shRNA constructs targeted Igl. One construct, Igl1 (272–300), specifically targeted Igl1. Three

constructs, Igl (1198–1226), Igl (2412–2440), and Igl (2777–2805), were targeted to sequences conserved in both Igl1 and 2 (Table 1). The GFP shRNA transfectants were LEE011 used as controls. Transfected trophozoites were selected with 100 μg/ml hygromycin for 48 hours before harvesting. Blots were probed with anti-Igl1 antibody, and with anti-actin antibody as a loading and normalization control. The level of Igl1 in the GFP shRNA transfectants was defined to be 100% (Figure 2, Table 4). The Igl1-specific (272–300) shRNA transfectant had a decreased amount of Igl1 protein, 27.8 ± 3.9%, as compared to the GFP shRNA control (Figure 2, Table 4). Igl (1198–1226) had 42.3 ± 6.2% and Igl (2777–2805) had 38.1 ± 9.4% of the GFP control Igl1 level. The Igl (2412–2440) Glutamate dehydrogenase shRNA construct had no effect on Igl1 levels (95.3 ± 9.7% of the level in the GFP shRNA transfectants)

(Figure 2, Table 4). HM1:IMSS nontransfected amebae were not statistically different from the GFP shRNA control (Table 4). The Igl (1198–1226) and Igl (2777–2805) transfectants, when selected with 30 μg/ml hygromycin rather than 100 μg/ml, yielded less knockdown, having ~70% and ~65% of the control level of Igl1 (data not shown). Table 4 Summary of Igl1 protein levels in Igl shRNA transfectants shRNA Transfectant or Control Sample % of Igl1 protein level (± SE) P-value GFP 100.0 ± 3.6   HM1:IMSS 115.5 ± 11.8 0.1449 Igl (2412–2440) 95.3 ± 3.2 0.2078 Igl1 (272–300) 27.8 ± 1.3 < 0.0001 Igl (1198–1226) 42.3 ± 2.1 < 0.0001 Igl (2777–2805) 38.1 ± 3.1 < 0.0001 The average level of Igl1 protein in the GFP control shRNA transfectants was defined as 100% expression of Igl1 protein for computational purposes.