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. 2024 Sep 30;7:1226. doi: 10.1038/s42003-024-06716-2

Fig. 5. Leishmania manipulates host CTR1 in multiple ways to reduce copper import.

Fig. 5

A Representative immunofluorescence image of CTR1 (green), co-stained with plasma membrane marker Na/K-ATPase (magenta), in J774A.1 macrophages with and without L. major infection (12 h) followed by basal, high copper (100 μM Cu) and copper chelated conditions (25 μM TTM) treatment for 2 h. The merged images represent colocalization of CTR1 with Na/K-ATPase. Both macrophage and Leishmania nuclei were stained with DAPI (blue). White arrows indicate intracellular parasites in infected cells (smaller nuclei). White arrowheads indicate endocystosed CTR1. The scale bar represents 5 μm. B Fraction of CTR1 colocalization with Na/K-ATPase from the above mentioned conditions demonstrated by a box plot with jitter points. The box represents the 25th to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5× interquartile range from the first and third quartiles. Asterisks indicate values that are significantly different from Uninf samples. Sample size of macrophage (n): 14, ∗∗∗p ≤ 0.001,∗∗∗∗p ≤ 0.0001, ns; (Wilcoxon rank-sum test). C Fraction of CTR1 colocalization with L.major compartments from the above mentioned conditions demonstrated by a box plot with jitter points. The box represents the 25th to 75th percentiles, and the median in the middle. The whiskers show the data points within the range of 1.5× interquartile range from the first and third quartiles. ns indicates values that are not significantly different from Inf samples. Sample size of macrophage (n): 14. ns non significant; (Wilcoxon rank-sum test). D Immunoblot of CTR1 of J774A.1 macrophages with or without infection (12 h) followed by indicated copper conditions. GAPDH is used as housekeeping control. E Immunoblot of CTR1 from infected and uninfected J774A.1 macrophages for 3 h with or without co-treatment with MG132 or Bafilomycin A1. The fold changes of CTR1 glycosylated monomer abundance normalized against housekeeping control GAPDH have been mentioned. F Immunoblot of CTR1 of J774A.1 macrophages after N-Glycosidase (PNGase 1 h) or O-glycosidase (O-Glycosidase & Neuraminidase Bundle 1 h) treatment. GAPDH is used as loading control. G Immunoblot of CTR1 from elute of biotin-streptavidin based pulldown of modified cysteine containing proteins from J774A.1 macrophages; and whole cell lystaes, with or without infection for 3 h. Fold changes of CTR1 in infected samples of the elute of the biotin pulldown normalized against uninfected control have been mentioned. GAPDH is used as housekeeping control for whole cell lysate.