Figure 1.

Binding of Fluorescent Propranolol (Propranolol-(β-Ala-β-Ala)-X-BODIPY630/650; Prop-BY630) to Triple-Negative Human Breast Cancer Cells (MDA-MB-231^HM cells)

(A) Fluorescence imaging of the binding of 50 nM Prop-BY630 to non-transfected MDA-MB-231^HM cells (upper panels) or MDA-MB-231^HM cells expressing NanoLuc-tagged human β₂-adrenoceptors (lower panels). Cells were pre-treated with Hoechst 33342 nuclear stain (2 μg/mL; blue labeling) and then labeled for 30 min with Prop-BY630 (red labeling). Upper right and bottom right panels show cells pre-incubated with 10 μM unlabelled propranolol before labeling with fluorescent propranolol (50 nM Prop-BY630). Cells were washed just before imaging to remove unbound fluorescent ligand. Data are representative images from 3 independent experiments. Scale bar represents 50μm.

(B) Bioluminescence imaging (Olympus LV200) of NanoLuc-tagged β₂-adrenoceptors. MDA-MB-231^HM Nluc-β₂AR cells treated with 400 nM furimazine substrate alone (upper panels) to detect luminescence in the absence of added fluorescent ligand using an open channel (20 s exposure time; 420 nm longpass filter; upper left panel) or a Cy5 channel (4 min exposure time; 600/50 nm bandpass filter; upper right panel) to detect BRET generated by binding of fluorescent ligand, when present. Middle and lower panels show images from cells treated with 50 nM Prop-BY630, in the presence (lower panels) or absence (middle panels) of unlabelled ICI 118551 (10 μM). Images shown were acquired with an open channel (middle and lower left panels) and the Cy5 channel (middle and lower right panels). Scale bar represents 50 μm.

(C) BRET ratios obtained using bioluminescence imaging using ImageJ time series analyzer. Data show the mean and SE obtained in 3 independent experiments. **p < 0.01 compared with basal or in the presence of 10 μM ICI 118551 (one-way ANOVA with Tukey's multiple comparisons).