Visualizing Autophagic Flux during Endothelial Injury with a Pathway-Inspired Tandem-Reaction Based Fluorogenic Probe

Autophagy is a dynamic and complicated catabolic process. Imaging autophagic flux can clearly advance knowledge of its pathophysiology significance. While the most common way autophagy is imaged relies on fluorescent protein-based probes, this method requires substantial genetic manipulation that severely restricts the application. Small fluorescent probes capable of tracking autophagic flux with good spatiotemporal resolution are highly demanable. Methods: In this study, we developed a small-molecule fluorogenic probe (AFG-1) that facilitates real-time imaging of autophagic flux in both intact cells and live mice. AFG-1 is inspired by the cascading nitrosative and acidic microenvironments evolving during autophagy. It operates over two sequential steps. In the first step, AFG-1 responds to the up-regulated peroxynitrite at the initiation of autophagy by its diphenylamino group being oxidatively dearylated to yield a daughter probe. In the second step, the daughter probe responds to the acidic autolysosomes at the late stage of autophagy by being protonated. Results: This pathway-dependent mechanism has been confirmed first by sequentially sensing ONOO- and acid in aqueous solution, and then by imaging autophagic flux in live cells. Furthermore, AFG-1 has been successfully applied to visualize autophagic flux in real-time in live mice following brain ischemic injury, justifying its robustness. Conclusion: Due to the specificity, easy operation, and the dynamic information yielded, AFG-1 should serve as a potential tool to explore the roles of autophagy under various pathological settings.

Anhydrous THF was distilled from Na prior to use. Reactions were monitored by thin layer chromatography using TLC Silica gel 60 F254 supplied by Qingdao Puke Seperation Meterial Corporation, Qingdao, P. R. China. Silica gel for column chromatography was 200-300 mesh and was supplied by Qingdao Marine Chemical Factory, Qingdao, P. R. China. Characterization of intermediates and final compounds was done using NMR spectroscopy and mass spectrometry.
NMR spectra were recorded on Brucker AVANCE III 400 NMR spectrometer or Brucker AVANCE III 500 NMR spectrometer or Bruker Ascend 600 NMR spectrometer with d-CHCl3 as solvent and tetramethylsilane (TMS) as the internal standard. The following abbreviations were used to designate multiplicities: s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet. All spectra were recorded at 25oC and chemical shifts were given in ppm and coupling constants (J) in Hz.
High-resolution mass data were obtained on an Agilent G6520 Q-TOF LC/MS.

Preparation of ONOO -
To a vigorously stirred solution of NaNO 2 (0.6 M, 10 mL) and H 2 O 2 (0.7 M, 10 mL) in

General experimental for photophysical property characterization
All the photophysical characterization experiments were carried out at an ambient temperature.
Absorption spectra were acquired using a Hitachi U-3010 spectrophotometer. Fluorescence measurements were performed on a Cary Elipse spectrofluorimeter with slit widths to be 2.5 and 5 nm for excitement and emission respectively except otherwise indicated, and the sensitivity of the instrument was kept at medium.
Deionized water was used to prepare all aqueous solutions. Phosphate buffer saline (PBS, 100 mM, pH 7.4) was purged with N 2 for 5 min before use. The probe was dissolved in EtOH to make a 50 μM stock solution.
To test the fluorescent responses of the probe towards ONOO -, aliquots of the probe stock solution were diluted with PBS and treated with ONOOto make sure both the probe and ONOOwere kept at desired final concentrations. After quick and vigorous shaking, the mixture was  Figure S1, which is corresponding to structure of AFG-1.

Supplementary Movies
Movie S1. Real-time visualization of autophagy using probe AFG-1 in ischemic living mice.
Real-time visualization of autophagy using in vivo two-photon laser scanning microscopy in live mice with brain microvessel injury. A combination of probe AFG-1 (green, λ ex 488 nm. λ em 505-550 nm) and adenovirus-mRFP-LC3 staining (red, λ ex 543 nm. λ em 560-615 nm) was used for fluorescent imaging. For two-photon imaging, 200 µm below the cortical surface were selected for imaging. The time-series images are individual frames from a continuous time-lapse movie and show dynamic AFG-1 and adenovirus-mRFP-LC3 fluorescence were elevation compared with the