Figure 4.
Figure 4. BAT1 acts as a pure antagonist against CXCL12, reducing migration along the chemokine gradient and cell viability in vitro. (A) Fluorescence microscopy analysis of receptor redistribution following exposure to CXCL12, BAT1, or plerixafor. Primary CLL cells were incubated with vehicle, 20 µM BAT1, or 20 µM plerixafor for 3 hours before exposure to CXCL12 for 10 minutes. Control cells were incubated with vehicle alone (no CXCL12). Cells were then stained for membrane and cytoplasmic CXCR4 to assess its distribution. Unstimulated CLL lymphocytes (top row) express CXCR4 at the cell surface and in the cytoplasm. Binding of CXCL12 causes receptor internalization with intracellular redistribution, with possible trafficking of cytoplasmic CXCR4 to the membrane (second row). Plerixafor (third row) and BAT1 (bottom row) bind to surface CXCR4, blocking immunoreactivity, but they do not induce receptor internalization or redistribution. Original magnification ×600 (60× 1.4 N.A. objective lens). Scale bars, 20 µm. (B) Immunoblotting demonstrates BAT1’s antagonism of CXCL12-induced signaling. Primary CLL cells were incubated with BAT1, plerixafor, or vehicle for 3 hours before exposure to CXCL12 for 10 minutes. Lysates were prepared, and signaling levels were assessed by immunoblotting for ERK phosphorylation (p-ERK). Incubation with BAT1 led to a dose-dependent reduction in p-ERK, with saturating levels of BAT1 reducing p-ERK to similar levels as 20 µM plerixafor or no stimulation. Densitometric quantification of the blots is shown with adjustment relative to total ERK for each lane. Percentage errors were calculated based on the average variation between equivalent blots across several experiments. (C) Effects of BAT1 and plerixafor on cell viability were assessed over 24 hours using flow cytometry. Primary CLL cells were incubated with plerixafor (i) or BAT1 (ii) at a range of concentrations over 24 hours. A progressive decrease in cellular viability was demonstrated at concentrations exceeding 20 nM BAT1 or plerixafor. Experiments were performed in triplicate, and the mean was taken. Errors are the standard deviation of the mean, added in quadrature to the standard error on the gating. Curves were fitted using nonlinear regression with software’s in-built log(agonist) vs response curve with variable slope, based on the Hill equation. (D) Inhibition of chemotaxis in response to CXCL12 was assessed using a filter migration assay, where cell migration was assessed using flow cytometry. BAT1 significantly reduces chemotactic migration of CLL lymphocytes. No significant difference in migration levels was seen between the BAT1-treated cells and those treated with plerixafor or vehicle alone. ***P < .001, 1-way analysis of variance with the Holm-Sidak multiple-comparisons test. ns, not significant.

BAT1 acts as a pure antagonist against CXCL12, reducing migration along the chemokine gradient and cell viability in vitro. (A) Fluorescence microscopy analysis of receptor redistribution following exposure to CXCL12, BAT1, or plerixafor. Primary CLL cells were incubated with vehicle, 20 µM BAT1, or 20 µM plerixafor for 3 hours before exposure to CXCL12 for 10 minutes. Control cells were incubated with vehicle alone (no CXCL12). Cells were then stained for membrane and cytoplasmic CXCR4 to assess its distribution. Unstimulated CLL lymphocytes (top row) express CXCR4 at the cell surface and in the cytoplasm. Binding of CXCL12 causes receptor internalization with intracellular redistribution, with possible trafficking of cytoplasmic CXCR4 to the membrane (second row). Plerixafor (third row) and BAT1 (bottom row) bind to surface CXCR4, blocking immunoreactivity, but they do not induce receptor internalization or redistribution. Original magnification ×600 (60× 1.4 N.A. objective lens). Scale bars, 20 µm. (B) Immunoblotting demonstrates BAT1’s antagonism of CXCL12-induced signaling. Primary CLL cells were incubated with BAT1, plerixafor, or vehicle for 3 hours before exposure to CXCL12 for 10 minutes. Lysates were prepared, and signaling levels were assessed by immunoblotting for ERK phosphorylation (p-ERK). Incubation with BAT1 led to a dose-dependent reduction in p-ERK, with saturating levels of BAT1 reducing p-ERK to similar levels as 20 µM plerixafor or no stimulation. Densitometric quantification of the blots is shown with adjustment relative to total ERK for each lane. Percentage errors were calculated based on the average variation between equivalent blots across several experiments. (C) Effects of BAT1 and plerixafor on cell viability were assessed over 24 hours using flow cytometry. Primary CLL cells were incubated with plerixafor (i) or BAT1 (ii) at a range of concentrations over 24 hours. A progressive decrease in cellular viability was demonstrated at concentrations exceeding 20 nM BAT1 or plerixafor. Experiments were performed in triplicate, and the mean was taken. Errors are the standard deviation of the mean, added in quadrature to the standard error on the gating. Curves were fitted using nonlinear regression with software’s in-built log(agonist) vs response curve with variable slope, based on the Hill equation. (D) Inhibition of chemotaxis in response to CXCL12 was assessed using a filter migration assay, where cell migration was assessed using flow cytometry. BAT1 significantly reduces chemotactic migration of CLL lymphocytes. No significant difference in migration levels was seen between the BAT1-treated cells and those treated with plerixafor or vehicle alone. ***P < .001, 1-way analysis of variance with the Holm-Sidak multiple-comparisons test. ns, not significant.

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