Fig. 5

Dose-dependent inhibition of N. caninum tachyzoites displayed by TAK-632 in vitro. A The inhibition curve of TAK-632 against N. caninum. The IC₅₀ was calculated using the log (inhibitor) versus normalized response-variable slope in GraphPad Prism (San Diego, CA, USA). B HFF cell viability upon treatment with TAK-632. The CC₅₀ for TAK-632 was calculated as 38.34 μM. C, D Plaque formation assay. 300 tachyzoites were inoculated into HFF cells. The infected cells were treated with different drugs. Cells were cultured for 9 days, and then fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. At least 10 plaques per well were randomly selected to measure the pixel size of each plaque using pixels in Photoshop CC software (Adobe, USA) to represent its plaque size. 1:0.1% DMSO; 2–5: 5, 2.5, 1.25, and 0.625 μM TAK-632; 6: 2.5 μM atovaquone; 7–10: HFF cells were not infected with tachyzoites and treated with 5, 2.5, 1.25 and 0.625 μM TAK-632, respectively. E Invasion assay. Extracellular tachyzoites were pretreated with different drugs for 3 h at 37 °C, followed by invasion for 1 h, and IFA was performed to determine the percentage of invaded parasites. F Intracellular proliferation assay. After infecting HFF cells with tachyzoites for 1 h, the infected cells were treated with different drugs. The proportion of vacuoles containing 2, 4, 8, and 16 tachyzoites was calculated by IFA. Results are shown as mean ± SD. Each assay was performed in triplicate. The results of plaque formation and invasion assays were analyzed by t-test. Intracellular proliferation assays were analyzed by two-way ANOVA in GraphPad Prism (San Diego, CA, USA). ****P < 0.0001; ***P < 0.001; **P < 0.01