Endoplasmic Reticulum Targeting Green Fluorescent Protein Chromophore-based Probe for the Detection of Viscosity

The occurrence of endoplasmic reticulum (ER) stress is the main cause of a variety of biological processes that are closely related to numerous diseases. The homeostasis of the ER microenvironment can be disrupted under ER stress. In this research, by linking a pentafluorophenyl to the green fluorescent protein chromophore, we have developed a new ER-targeting fluorescent probe (GE-Y) for measuring changes of intracellular ER viscosity caused by ER stress. Importantly, an increase in ER viscosity was observed using GE-Y in cells undergoing autophagy. As such, our research provides an ideal tool for studying ER stress and autophagy.


Optical Measurements
In a typical optical test, GE-Y (10 μΜ) solutions were obtained by diluting the probe stock solution (1 mM) into a PBS/glycerin system of different viscosities (from 0.89 cP to 438.40 cP at 25°C). The volume ratio of PBS buffer (10 mM, pH 7.4) and glycerin of different viscosity solutions is shown in Table S1. All the absorption and emission spectra were recorded using standard 1-cm path length quartz fluorescence cuvettes at 25°C. The slit width of excitation and emission were set to 10 nm and excitation wavelength was set at 430 nm for the fluorescence spectroscopy.

Cell Culture and cell viability
The human breast cancer (MCF-7) cells, human cervical cancer cells (HeLa), human alveolar epithelial cells (A549), and Chinese hamster ovary cells (CHO) were purchased from the Cell Bank of the Chinese Academy of Sciences. Cells were incubated in medium (high glucose DMEM for MCF-7 cells and HeLa cells, F-12 for A549 cells and CHO cells) supplemented with 10% FBS (fetal bovine serum) and placed in a 37°C constant temperature incubator containing 5% carbon dioxide. The toxicity of GE-Y towards MCF-7 cells was evaluated by the standard MTT method. Firstly, the cells were seeded in a 96-well cell culture plate at a density of 8000 cells/well with medium volume of 200 μL.
Then, the plate was placed in a 37°C constant temperature incubator under 5% CO2 for 12 h. Subsequently, probe GE-Y of different concentrations (0, 4, 8, 12, 16, 20 μM) were added to the wells and five parallel groups were set. After the culture medium was removed, 100 μL of 3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2-H-tetrazolium bromide (MTT) reagent (1 mg/mL) was loaded in to each well after culturing the cell for 12 h and 24 h, respectively, followed by incubation at 37 °C for another 4 h. Then the MTT reagent was removed and 100 μL DMSO was added to each well. The plate was placed on an oscillator and S5 gently shaken for 10 minutes to dissolve the blue crystals, and the optical density value was measured using a Thermo scientific multiskan spectrometer.

Western Blot Assay
The treated cells were washed twice with PBS buffer. After incubating with 100 μL RIPA lysis buffer at 4℃ for 10 min, the supernatant was collected by centrifuging at 12 000 rpm for 5 minutes. Total protein of supernatant was determined by BCA protein quantitative kit. 30 μg total protein was separated by 12% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% BSA at 25℃ for 2h and incubated with primary rabbit polyclonal LC3 (1:1000) at 4°C overnight with gentle shaking. Washing membranes three times with TBST buffer, and incubated with HRP-conjugated secondary antibodies (1:2000) for 1 hour at 25 ℃. After washing with TBST buffer three times, the membranes were incubated with ECL chemiluminescence detection reagent and exposed to X-ray film.
Protein light chain 3(LC3) is a biomarker of autophagy. 1 When autophagy occurs, LC3 precursor molecules are cleaved to form cytoplasmic LC3-Ⅰ by removing the c-terminal 5-peptide. Subsequently, LC3-Ⅰ was coupled with phosphatidylethanolamine to form membrane-bound LC3-Ⅱ, so the ratio of LC3-Ⅱ/LC3-Ⅰ could be used to evaluate the degree of autophagy. We monitored the ratio of LC3-Ⅱ/LC3-Ⅰ by western blot to ensure autophagy occurred in the experimental group.

Synthetic procedures
Scheme S1. Synthetic route for preparing GE-Y.

Synthesis of HBDI
AcBDA (100 mg, 0.4 mmol) and methylamino alcohol (4 mL) were added to a 50 mL S8 round bottom flask. The mixture was stirred at 25°C under nitrogen for 2-3 hours. Then the solvent was removed under reduced pressure. Pyridine (5 mL) was added to dissolve the residue and the mixture continued to reflux at 110°C under nitrogen for 4-5 hours.
After removal of the solvent, the residue was purified by flash chromatography on silica gel using CH2Cl2/CH3OH (v/v, 110/1) as eluent to afford HBDI as a yellow solid (71 mg, 80%). 1

Fluorescence quantum yield for GE-Y
The       Table S3. Gray value of LC3-Ⅰ and LC3-Ⅱ protein in live MCF-7 cells in Figure S9. The gray values were obtained by using the ImageJ software. Figure S16 13 C NMR spectra of GE-Y.