Fluorescence live cell imaging revealed wogonin targets mitochondria

Scutellaria baicalensis is one of the widely used Chinese traditional medicines, and wogonin is one of major active components in it. However, the mechanism of action of wogonin has largely remained unclear. In this work, we designed a fluorescent probe, namely ATTO565-WGN, by conjugating wogonin with the fluorophore ATTO565 based on Mannich reaction via a flexible chain linker. In vitro assays verified that the ATTO565-WGN conjugate has a similar anti-proliferative activity to wogonin against human A549 and HeLa cancer cell lines. Combining co-localization and competition studies, confocal fluorescence imaging clearly demonstrated that the fluorescent wogonin probe predominantly located in mitochondrial area of living cells, indicating that wogonin acts at mitochondrion to exert its pharmacological functions. Significantly, the conjugated ATTO565 fluorophore conferred the wogonin probe STED (Stimulated Emission Depletion) feature, enabling STED fluorescence living cell imaging with a 55 nm of ultrahigh spatial resolution. This will greatly beneficial for the in situ investigation of interactions between wogonin and biological targets at the finely organized and dynamic mitochondria of living cells. Moreover, this work also provides novel insights into rational design of mitochondrion targeting fluorescence probes for ultrahigh resolution living cell imaging.