Leucine aminopeptidase may contribute to the intrinsic resistance of cancer cells toward cisplatin as revealed by an ultrasensitive fluorescent probe† †Electronic supplementary information (ESI) available: Experimental section and supporting figures. See DOI: 10.1039/c5sc03600c Click here for additional data file.

Leucine aminopeptidase may contribute to the intrinsic resistance of cancer cells toward cisplatin as revealed by an ultrasensitive fluorescent probe.

LTD, Shanghai, China. Radio-Immunoprecipitation Assay (RIPA) Lysis Buffer was purchased from CWbiotech. Co. LTD, Beijing, China. Proteins were pure as judged by Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Fetal bovine serum (FBS) was obtained from ExCell Biology, USA. Anti-LAP antibody was purchased from proteintech TM , USA. All other chemicals used were local products of analytical grade.
Ultrapure water (over 18 MΩ·cm) from a Milli-Q reference system (Millipore) was used throughout. The stock solution (1.0 mM) of probe 1 was prepared by dissolving requisite amount of it in DMSO. Stock solutions of other substances were prepared by dissolving in PBS or water.
Detection of LAP in human liver microsomes. In a test tube, 500 μL of human liver microsomes (1 mg/mL) and appropriate volume of PBS were mixed, followed by addition of an appropriate volume of ethanol. The mixed solution was adjusted to 2 mL with PBS, and the final concentration of human liver microsomes was 0.25 mg/mL. After incubation at 37 °C for 1 h, 20 μL of stock solution of probe 1 was added and the mixed solution was incubated 37 °C for additional 25 min. Finally, 2 mL of the reaction solution was transferred to a quartz cell of 1-cm optical length to measure fluorescence with λ ex = 585 nm (both excitation and emission slit widths were set to 10 nm). Data are expressed as mean ± standard deviation (SD) of three separate measurements.
Cell imaging. Cells (HepG2 or A549) were treated at 37 °C for different periods of time (0 -24 h) with various concentrations of cisplatin (0 -2 mg/L) in Petri dishes.
Before cell imaging, the culture media were removed, and the cells were washed using DMEM for three times. Then, the cells were incubated with the probe (10 μM) at 37 °C for 20 min in DMEM, washed with DMEM to remove the free probe, and subjected to fluorescence imaging experiments. Unless otherwise noted, data are expressed as mean ± standard deviation (SD) of three separate measurements.
LAP assay by ELISA kit. The microsomes (0.25 mg/mL) were used directly. The cell lysate was prepared according to the following procedure. In a test tube, 1.210 5 cells (HepG2 or A549) in 1 mL DMEM was centrifugated at 3000 rpm for 5 min, and then the supernatant was discarded, followed by washing the cells with PBS for three times. After discarding the PBS, 200 μL RIPA Lysis Buffer was added to the test tube, and the tube was left at 0 °C for 20 min. Finally, the tube was centrifugated at 3000 rpm for 10 min, and the supernatant was collected for use. The concentration of LAP in microsomes or cells was determined by measuring the absorbance at 450 nm using a commercial LAP ELISA kit. Briefly, to each well, 40 μL samples or 50 μL standards, 10 μL biotin tagged LAP antibody, and 50 μL enzyme marked reagents were mixed. After incubating the reaction mixture at 37 °C for 60 min, the wells were washed five times with wash solution, and then 50 μL chromogen reagents A and B were added in the wells in succession, followed by incubating for 10 min at 37 °C. Finally, 50 μL stop buffer was added to the wells, and the absorbance of each well was recorded at 450 nm on a Microtiter Plate assay system. The standard curve for LAP detection was constructed following the direction of the kit in the concentration range from 0 to 100 ng/mL, and the corresponding concentrations were calculated according to the standard curve. Data are expressed as mean ± standard deviation (SD) of five separate measurements.
Cytotoxicity assay. Unless otherwise noted, the cytotoxicity of probe 1 or cisplatin was evaluated by the standard MTT assay. Briefly, cells (HepG2 or A549) were seeded in 96-well U-bottom plates at a density of 7000 cells/well, and incubated with probe 1 or cisplatin at varied concentrations at 37 °C for 24 h (72 h for IC 50 determination). Then, the culture media were discarded, and 0.1 mL of MTT solution (0.5 mg/mL in DMEM) was added to each well, followed by incubation at 37 °C for 4 h. The supernatant was abandoned, and 0.11 mL of DMSO was added to each well to dissolve the formed formazan. After shaking the plates for 10 min, absorbance values of the wells were read with a Microtiter plate assay system at 490 nm. The cell viability rate (V R ) was calculated according to the equation: V R = A/A 0 × 100 %, where A is the absorbance of the experimental group (the cells treated by probe 1 or cisplatin) and A 0 is the absorbance of the control group (the cells untreated by probe 1 or cisplatin). Data are expressed as mean ± standard deviation (SD) of five separate measurements.  Cancer, 2006, 118, 1390).
Western blot. The HepG2 and siRNA-transfected HepG2 cells were lysed with RIPA buffer, and the cell lysates were diluted with PBS to obtain a solution of about 0.5 mg/mL of total proteins. At the same time, the protein standard solution of 0.5 mg/mL was prepared. The protein concentrations and Western blot analysis were performed according to the literatures (Kondo et al. Int. J. Cancer, 2006, 118, 1390Fang et al. J. Am. Chem. Soc. 2014, 136, 226;Li et al. J. Exp. Bot. 2011, 62, 4763).

Statistical tests.
The t values were calculated from formula 1 below, where X 1 and X 2 were the averages of the two samples, n 1 and n 2 were sample capacities, and S 1 and S 2 were standard deviations of the two samples, respectively. The two-sided Student's t-test was used to evaluate the significant difference between the data.      Figure S5. ESI mass spectrum of the reaction products of 1 with LAP.