Generation of <i>TcIPCS</i> addback cell lines, protein localization and enzyme activity.

(A) Schematic representation of the linearized TcIPCS-HA expression vector used for transfection and integration in the tubulin locus to generate the addback line. (B) PCR analysis of WT, TcIPCS KO cell line (KO8) and two cloned addback cell lines (AB1 and AB3) obtained after transfection of the TcIPCS KO8 clone with the TcIPCS-HA expression vector. Annealing positions of the primers and the expected sizes of the amplicons are indicated in the schematic representation of one wild type TcIPCS allele (top figure), one disrupted TcIPCS allele (medium) and in the addback sequence inserted in the tubulin locus (bottom). Agarose gel electrophoresis analyses showed PCR products obtained with DNA extracted from WT epimastigotes, the TcIPCS KO8 cell lines and the two cloned addback cell lines AB1 and AB3. (C) Relative expression levels of TcIPCS transcripts quantified by RT-qPCR using RNA extracted from WT, from the TcIPCS KO cell line KO8 and from the addback cell line AB1. (D) Western blot analysis with anti-HA antibody of HA-tagged TcIPCS and total protein extracts from WT, TcIPCS KO cell lines KO4 and KO8, a transfected population derived from the KO8 cell line (pop. addback) and a transfected population derived from WT epimastigotes (pop super-expressor). The bands corresponding to the HA-tagged TcIPCS protein with a MW of 37 KDa are indicated. (E-H) Fluorescence microscopy analyses of epimastigotes incubated with NBD C6-Ceramide to stain ceramide rich regions (red), such as the Golgi apparatus (E). Parasites showing the immunostaining using anti-HA antibody (green) indicate TclPCS protein localization (F). Merged image showing the co-localization of the protein with ceramide-rich regions, close to the kinetoplast, indicates that TcIPCS localizes to the Golgi apparatus (G). White arrows indicate co-localization of the TclPCS protein with NBD C6-ceramide close, but not at the same location to the parasite kinetoplast, where the Golgi apparatus is localized (H). (I) HP-TLC analysis of IPC present in extracts from T. cruzi epimastigotes, Vero cells (negative control) and L. major (positive control). (J) HP-TLC analysis of IPC present in T. cruzi epimastigotes extracts derived from WT, TcIPCS KO4 and KO8 cell lines as well as in the TcIPCS addback cell line AB1. In I and J, cells were incubated in presence of fluorescent ceramide and immediately used for preparation of lipid extracts. The migration positions of fluorescent inositol phosphorylceramide (IPC), sphingomyelin (SM) as well as the origin (O) and the front (F) are indicated. The presence of an unknown putative lipid was also identified.