Characterization of Δ<i>kh1</i> axenic amastigotes.

<p>A. Immunoblot of wild type (wt) and <b>Δ</b><i>kh1</i> mutant probed with anti-P4 and anti-<b>α</b>-tubulin antibodies as a loading control. The positions of molecular weight markers are indicated. B. Immunofluorescence of the P4 amastigote-specific antigen. Left, axenic amastigotes were stained with anti-P4 antibody (P4, green), DAPI (blue), and anti-<b>α</b>-tubulin antibody (Tub, red). The 100% shown in the P4 panel represents the percentage of cells analyzed that are positive for P4 (wt, n = 212; <b>Δ</b><i>kh1</i>, n = 240). Right, promastigotes were stained for the same marker as a negative control for the P4 antibody. Scale bar, 3 <b>μ</b>m. C. Growth curve of axenic amastigotes comparing growth rate of wild type, <b>Δ</b><i>kh1</i>mutants, and <b>Δ</b><i>kh1</i>[int<i>Kh1</i>] add-back lines over a 16-day time course. Numbers represent the averages and ranges from two independent growth curves (n = 2). Each sample from each growth curve time point was counted in triplicate and the values averaged. D. Quantification of infection of THP-1 macrophages with wild type, <b>Δ</b><i>kh1</i>mutants, and <b>Δ</b><i>kh1</i>[int<i>Kh1</i>] add-back axenic amastigotes. Times post infection: h = hours, D = days. Data represent the averages and standard deviations of triplicate infections (n = 3), each counted in triplicate and averaged. This experiment is similar to that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134432#pone.0134432.g002" target="_blank">Fig 2E</a>, except that axenic amastigotes, rather than promastigotes, were employed to initiate infection.