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. 2015 Mar 13;9(3):e0003588. doi: 10.1371/journal.pntd.0003588

Fig 1. Modulation of CYP51 levels in L. donovani.

Fig 1

A, Replacement of a CYP51 allele by homologous recombination. Correct targeting of the knockout cassettes was verified by PCR using one primer within the knockout cassette and one upstream of CYP51 (primers 7 and 8 (hygromycin), top or 7 and 9 (puromycin), bottom). B, qPCR quantification of chromosomal CYP51 levels, normalized to SAT or to CBS and to wild-type levels. C, CYP51 protein levels in half knockout and complemented strains. CYP51 and GAPDH were detected by Western blot (top) and quantified by densitometry (bottom) D, In vitro infectivity of half knockout and complemented strains. THP1 macrophages were infected at a 10:1 parasite to macrophage ratio. Cells were fixed and stained with DAPI 24, 48 and 72 h post-infection, and macrophage infection levels were determined by automated high-throughput imaging and parasite detection. E, In vivo infectivity of half knockout and complemented strains. BALB/c mice were infected intravenously. Liver parasite burden (Leishman-Donovan Units, LDU) was determined 28 days post-infection by counting stained liver impressions. F, Sterol profiling by GC-MS.