Pyroglutamate aminopeptidase 1 may be an indicator of cellular inflammatory response as revealed using a sensitive long-wavelength fluorescent probe† †Electronic supplementary information (ESI) available: Experimental section and supporting figures. See DOI: 10.1039/c6sc00951d

Pyroglutamate aminopeptidase 1 may be an indicator of cellular inflammatory response as revealed using a sensitive long-wavelength fluorescent probe.

amount of it in DMSO. Stock solutions of other substances were prepared by dissolving in PBS or water.
Apparatus. Fluorescence measurements were made on a Hitachi F-4600 fluorimeter (Tokyo, Japan). UV-vis absorption spectra were measured in 11 cm quartz cells with a TU-1900 spectrophotometer (Beijing, China). A model HI-98128 pH-meter (Hanna Instruments Inc., USA) was used for pH measurements. 1 H NMR and 13 C NMR spectra were measured with a Bruker Avance 400 or 600 spectrometer in CD 3 OD or DMSO-d 6 . Electrospray ionization mass spectra (ESI-MS) were measured with an LC-MS 2010A (Shimadzu) instrument. High resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an APEX IV FTMS instrument (Bruker, Daltonics). Cell imaging experiments were operated on a FV 1000-IX81 confocal laser scanning microscope (Olympus, Japan). The fluorescence quantum yield (Φ) was determined by using fluorescein (Φ = 0.85 in 0.1 M NaOH) as a standard (Zhang et al, J. Am. Chem. Soc. 2011, 133, 14109). The western blot signal was detected using an ECL kit. Image processing was made with Olympus ImageJ software (National Institutes of Health, USA). Cytotoxicity assay was made on microtiter plate assay system (Molecular Devices, USA).
Cell imaging. Cells (LO-2, HepG2 or RAW264.7) were treated at 37 °C for different periods of time (0-24 h or 0-16 h) with various concentrations of FIA (0-0.25%, v/v) or LPS (0-1.0 g/mL). For inhibitor experiments, cells were treated according to the above method and then incubated with iodoacetamide (5 nM and 50 nM) in Petri dishes. Before cell imaging, the culture media were removed, and the cells were washed using DMEM or RPMI-1640 for three times. Then, the cells were incubated with the probe (5 μM) at 37 °C for 30 min in DMEM or RPMI-1640, washed with DMEM or RPMI-1640 to remove the free probe, and finally subjected to fluorescence imaging. Unless otherwise noted, data are expressed as mean  standard deviation (SD) of three separate measurements.
Cytotoxicity assay. The cytotoxicity of probe 1 was tested on LO-2 cells using a standard MTT assay, as described previously (Gong et al, Chem. Sci. 2016, 7, 788).
Preparation of cell lysates. The cell lysates were prepared according to our previous method (Gong et al, Chem. Sci. 2016, 7, 788).
Western blot. Unless otherwise stated, all the western blot analyses were made by using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a protein standard. In brief, cells (LO-2 or RAW264.7, including control group, and FIA, LPS and inhibitor treated groups) were lysed with RIPA buffer, and the cell lysates were diluted 10 times with PBS to obtain a solution of about 0.5 mg/mL of total proteins. The diluted cell lysates were incubated with Anti-PGP-1 or Anti-TNF- antibody for 3 h at room temperature, and then incubated with Protein A/G Agarose for 1.5 h at room temperature. The agarose beads were pelleted by centrifugation at 4 °C (14000 rpm for 5 min), and the PGP-1 or TNF--depleted supernatant was collected and preserved at -70 °C. For western blot analysis, equal amounts of protein in cell lysates were separated by SDS-PAGE and electroblotted onto PVDF membranes. After blocking with 5% skim milk in TBST (20 mM Tris, pH 7.6, 140 mM NaCl, and 0.1% Tween-20) at room temperature for 1.5 h, the membranes were incubated with the corresponding primary antibodies at 4 °C overnight. The membranes were washed with TBST for three times and further incubated with the horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h. Finally, the signal was detected using an ECL kit. The grayscale value was analyzed by Gel-Pro32 software.   The effects of iodoacetamide at varied concentrations on the fluorescence intensity of (1) probe 1 (5 μM) and (2) cresyl violet (5 μM). The reactions were performed at 37 °C for 30 min in 6.7 mM PBS (pH 7.4). λ ex/em = 585/625 nm. As is seen, the presence of iodoacetamide does not affect the fluorescence of probe 1 and its reaction product.  The HepG2 cells were pretreated with 0.25% FIA, then with 50 nM iodoacetamide for 1 h, and finally incubated with the probe (5 μM) as above. (B2-E2) The HepG2 cells were pretreated with LPS at varied concentrations (0, 0.2, 0.5, 1.0 μg/mL, respectively) for 16 h, and then incubated with the probe (5 μM) for 30 min. (F2) The HepG2 cells were pretreated with 1.0 μg/mL LPS, then with 50 nM iodoacetamide for 1 h, and finally incubated with the probe (5 μM) as above. (G1, G2) Relative pixel intensities measurements (n = 3) from the above corresponding images by using software ImageJ (the pixel intensities from images E1 and E2 are defined as 1.0). Scale bar, 20 μm.