Fig. 4: Impact of Crizotinib on ROS1 Expression and Cell Viability in T. annulata-Infected Cells.

a Immunofluorescence analysis showing ROS1 expression in TA1 cells. Cells were stained with anti-ROS1 (magenta) and DAPI for nuclear staining (blue). Brightfield image is included for reference. The presented image is a maximum intensity projection (MIP) of a Z-stack acquired using fluorescence microscopy. b Western blot analysis showing ROS1 expression (50–80 kDa) in healthy peripheral blood mononuclear cells (hPBMC) and T. annulata-infected cell lines (TA1, TA3, and TA4). β-actin (43 kDa) was used as a loading control. The blot shown is representative of three independent experiments (n = 3). c Bar graph representing the IC50 values of crizotinib in four T. annulata-infected cell lines (TA1, TA2, TA3, and TA4). Data are derived from three biological replicates, and error bars indicate the standard error of the mean (SEM). The mean IC50 value for each cell line is displayed above the corresponding bar. d Bar graph representing the percentage of cell death in T. annulata-infected cells following crizotinib treatment at 24 and 48 h. Untreated controls for each time point are included for comparison. Statistical analysis was performed between treated and corresponding control groups at each time point. Data are presented as mean ± SEM from 3 independent experiments. e Western blot analysis of ROS1 expression in the TA cell line following crizotinib treatment for 24 and 48 hours. Cell lysates from treated and untreated (control) samples were collected and probed for ROS1. β-actin was used as a loading control. f Densitometric quantification of ROS1 expression from two biological replicates, normalized to β-actin. Data are represented as a bar graph comparing treated samples to control at each time point. g qPCR analysis of ROS1, cMET, and ALK1 expression in T. annulata infected cells. Data are represented as log2 fold change (FC) relative to healthy PBMCs, which were used as the non-infected control. The experiments were performed with three biological replicates, and HPRT was used as the housekeeping control.