Figure 2.
P5-C mimics biological activity of P5
(A) P5-C mimics biological activity of P5 in human prostate cancer and glioma cell lines
The LNCaP (top panel) and U87MG (bottom panel) cells were incubated equivalent amounts of P5-C (right histograms) and P5 (left histogram). Cell proliferation was evaluated using the XTT cell viability assay in at least five independent experiments. Statistical test were done using Student's t test; ∗p < 0.05, ∗∗p < 0.01. Data are represented as mean ± SEM.
(B) P5-C mimics biological activity of P5 in mouse primary T cells and inhibits T cell proliferation. Naive CD4+ T cells were stained with CellTrace Violet and activated under Th2 differentiation conditions for 72 h in the presence (red histogram) or absence (blue histogram) of 5 μM P5 or P5-C. The cell proliferation profile was captured by a flow-cytometry-based dye decay assay. Data shown are representative of three independent experiments with three mice in each experiment.
(C) P5-C mimics biological activity of P5 in mouse primary B cells and inhibits B cell immunoglobulin class switching. Naive resting B cells were induced with LPS and IL-4 in the presence of different concentrations of P5 and P5-C (0, 5, and 10 μM). Cell-surface expression of IgG1 was analyzed by flow cytometry on the fifth day of stimulation. Data shown are representative of three independent experiments with three mice in each experiment.
(D) The P5-C probe mimics P5 interaction with the previously known P5-binding protein CLIP1 and is competed out by cold P5. HA-tagged CLIP1 was ectopically expressed in HEK cells, and the protein content of the lysate was estimated. About 400 μg of whole-cell lysate was used in two experiments simultaneously, one incubated with 50 nM P5-C and another with 500 nM P5 before 50 nM P5-C. The azide-coated magnetic beads were clicked and pulled down using magnets. The magnetic-azide beads were incubated with cell lysate to capture any background binding of CLIP1 to the beads. SDS-PAGE and western blotting followed by incubation with HA antibody revealed P5-C binding to CLIP 1, and P5 competed out P5-C binding.
(E) P5-C is cell permeable. Live LNCaP cells are incubated with P5-C, which is clicked to Alexa Fluor 488 azide (in green) and imaged under a fluorescence microscope. Simultaneously, as a control, cells not incubated with P5-C were clicked and imaged as before. DAPI (in blue) was used in both the experiments to visualize the nucleus. Scale bar: 50