Supporting Information for:

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1 Supporting Information for: Ultrasensitive Fluorescent Probes Reveal an dverse ction of Dipeptide Peptidase IV and Fibroblast ctivation Protein during Proliferation of Cancer Cells Qiuyu Gong,, Wen Shi, Lihong Li, Xiaofeng Wu and Huimin Ma*,, eijing ational Laboratory for Molecular Sciences, Key Laboratory of nalytical Chemistry for Living iosystems, Institute of Chemistry, Chinese cademy of Sciences, eijing 9, China. * phone: ; fax: University of Chinese cademy of Sciences, eijing 49, China Table of Contents Figure S. H MR and 3 C MR spectra of probe. Figure S. The HR-ESI-mass spectra of probe. Figure S3. H MR and 3 C MR spectra of probe. Figure S4. The HR-ESI-mass spectra of probe. Figure S5. ESI mass spectra of the reaction products. Figure S6. The inhibitor experiments of the reaction systems. Figure S7. Effects of reaction temperature and ph on the fluorescence of reaction systems. Figure S8. Effects of reaction time on the fluorescence of reaction systems. Figure S9. The plots of fluorescence enhancements of commercial probes versus the enzymes concentrations. Figure S. Fluorescence change of probe in the presence of various species. Figure S. Fluorescence change of probe in the presence of various species. Figure S. The influence of enzymes on each other. Figure S3. The Michaelis-Menten equations of the reaction systems. S-

2 Figure S4. Effects of probes at varied concentrations on the cell viability of MGC83 cells. Figure S5. The changes of DPPIV and FP in H89PM cells with varied genistein. Figure S6. Representative IHC images of H89PM cells. Figure S7. Fluorescence images of MK8 cells. Figure S8. Fluorescence images of MGC83 cells. Figure S9. Fluorescence images of H89PM cells. Figure S. Effects of genistein at varied concentrations on the fluorescence of reaction systems. Figure S. The relationship between enzymes concentrations and cell viabilities. Figure S. Western blot analysis of normal and sir-transfected MGC83 cells. S-

3 H H C H CH 3 C - H H C H CH 3 C - Figure S. H MR and 3 C MR spectra of probe. () H MR (4 MHz, CD 3 D, 98 K) and () 3 C MR ( MHz, CD 3 D, 98 K) spectra of probe. Figure S. The HR-ESI-mass spectra of probe. S-3

4 H H H CF3C - H H H CF 3 C - Figure S3. H MR and 3 C MR spectra of probe. () H MR (6 MHz, CD 3 D, 98 K) and () 3 C MR ( MHz, CD 3 D, 98 K) spectra of probe. Figure S4. The HR-ESI-mass spectra of probe. S-4

5 8. 7. Inten. (x,) H H m/z.5.5. Inten. (x,,) H H m/z Figure S5. ESI mass spectra of the reaction products. () with FP and () with DPPIV Wavelength (nm) 6 4 PT- ( M) C 6 D 6 E W avelength (nm) [Vildagliptin] (nm) [Linagliptin] (nm ) Figure S6. Inhibitor experiments. () Fluorescence spectra of probe in different reaction systems: () probe only (5 μm); () probe (5 μm) + FP (.6 μg/ml); (3) system + 5 μm PT-. () The effects of PT- at varied concentrations on the fluorescence intensity of () probe (5 μm) and () cresyl violet (5 μm). (C) Fluorescence spectra of probe in different reaction systems: () probe only ( μm); () probe ( μm) + DPPIV (. μg/ml); (3) system + nm vildagliptin; (4) system + nm linagliptin. The effects of (D) vildagliptin and (E) linagliptin at varied concentrations on the fluorescence intensity of () probe ( μm) and () cresyl violet ( μm). Linagliptin was chosen for cell experiments below. The reactions were performed at 37 C in PS (ph 7.4). λ ex/em = 585/65 nm. S-5

6 4 3 probe + FP 4 3 probe +FP F F probe Temperature ( C) probe ph 6 C probe +DPP4 6 D probe +DPP4 F 8 F 8 4 probe Temperature ( C) 4 probe ph Figure S7. Effects of reaction temperature and ph on the fluorescence of reaction systems. Effects of reaction temperature () and ph () on the fluorescence of probe (5 μm) in the absence and presence of FP (.6 μg/ml). Conditions: the reaction was performed in PS (ph 7.4) for 3 h () at different temperatures and () at different ph values adjusted with dilute HCl or ah. Effects of reaction temperature (C) and ph (D) on the fluorescence of probe ( μm) in the absence and presence of DPPIV (. μg/ml). Conditions: the reaction was performed in PS (ph 7.4) for 3 min (c) at different temperatures and (d) at different ph values adjusted with dilute HCl or ah. λ ex/em = 585/65 nm. From this figure it can be seen that DPPIV and FP function well under physiological conditions (ph 7.4 and 37 C). F 45 9 ng/ml Time (min) F ng/ml Time (min) Figure S8. Effects of reaction time on the fluorescence of reaction systems. () plot of fluorescence enhancement of probe (5 μm) versus the reaction time in the presence of varied FP concentrations. () plot of fluorescence enhancement of probe ( μm) versus the reaction time in the presence of varied DPPIV concentrations. The reaction was performed at 37 C in PS (ph 7.4). λ ex/em = 585/65 nm. s seen, a reaction time of 3 h may be chosen for FP, and 3 min for DPPIV. S-6

7 F C (ng/ml) F C (ng/ml) Figure S9. The plots of fluorescence enhancements of commercial probes versus the enzymes concentrations. () plot of fluorescence enhancement of commercial FP probe (5 μm) versus the FP concentration. The reactions were performed at 37 C in PS (ph 7.4) for 3 h; λ ex/em = 34/44 nm. () plot of fluorescence enhancement of commercial DPPIV probe ( μm) versus the DPPIV concentration. The reactions were performed at 37 C in PS (ph 7.4) for 3 min; λ ex/em = 34/44 nm. The detection limits of the commercial probes were determined to be 9.8 ng/ml FP and.35 ng/ml DPPIV. Figure S. Fluorescence change of probe in the presence of various species. () probe only (5 μm; control); () 5 mm KCl; (3).5 mm CaCl ; (4) μm ZnCl ; (5).5 mm MgCl ; (6) μm CuS 4 ; (7) mm glucose; (8) mm vitamin C; (9) mm glutamic acid; () mm cysteine; () 5 mm glutathione; () μm Cl ; (3) μm human serum albumin; (4) 4 ng/ml LP; (5) μg/ml V8 protease; (6) μg/ml prolidase; (7) μg/ml esterase; (8) μg/ml trypsin; (9) μg/ml thrombin; () μg/ml MMP-; ().9 μg/ml DPPIV; ().6 μg/ml FP. S-7

8 Figure S. Fluorescence change of probe in the presence of various species. () probe onlyonly ( μm; control); () 5 mm KCl; (3).5 mm CaCl ; (4) μm ZnCl ; (5).5 mm MgCl ; (6) μm CuS 4 ; (7) mm glucose; (8) mm vitamin C; (9) mm glutamic acid; () mm cysteine; () 5 mm glutathione; () μm Cl ; (3) μm human serum albumin; (4) 4 ng/ml LP; (5) μg/ml V8 protease; (6) μg/ml prolidase; (7) μg/ml esterase; (8) μg/ml trypsin; (9) μg/ml thrombin; () μg/ml PGP-; () μg/ml MMP-; ().8 μg/ml FP; (3). μg/ml DPPIV Figure S. The influence of enzymes on each other. () The influence of DPPIV at varied concentrations on FP. () Probe (5 μm) +.6 μg/ml FP; () system +.6 μg/ml DPPIV; (3) system +. μg/ml DPPIV; (4) system +.4 μg/ml DPPIV. The reactions were performed at 37 C in PS (ph 7.4) for 3 h. () The influence of FP at varied concentrations on DPPIV. () Probe ( μm) +. μg/ml DPPIV; () system +. μg/ml FP; (3) system +.4 μg/ml FP; (4) system +.8 μg/ml FP. The reactions were performed at 37 C in PS (ph 7.4) for 3 min. λ ex/em = 585/65 nm. s seen, the two enzymes scarcely interfere with each other. S-8

9 /V (min M - ) K m = M V max =.8 M/min /[probe] ( M - ) /V (s M - ) 4 3 K m = M V max =.3 M/s /[probe] ( M - ) Figure S3. The Michaelis-Menten equations of the reaction systems. The Michaelis-Menten equation was described as: V = V max [probe]/(k m +[probe]), where V is the reaction rate, [probe] is the probe concentration, and K m is the Michaelis constant. Conditions: () 6 ng/ml FP,.5-4 μm probe. () 3 ng/ml DPPIV,.5-3 μm probe. ph 7.4, temperature 37 C. λ ex/em = 585/65 nm. Cell Viability (%) probe ( M) 8 4 Cell Viability (%).5 5 Probe ( M) Figure S4. Effects of probes at varied concentrations on the cell viability of MGC83 cells. Effects of () probe and () probe at varied concentrations on the cell viability of MGC83 cells. The cell viability without the probe is defined as %. C D E C D E F * C D E DPPIV/GPDH G DPPIV GPDH C D E F C D E FP/GPDH G FP GPDH.S. C D E Figure S5. The changes of DPPIV and FP in H89PM cells with varied genistein. () H89PM cells only; (-E) the cells were pretreated at 37 C for 4 h with genistein at S-9

10 varied concentrations (, 5,, μg/ml, respectively), and then incubated with probe ( μm). () H89PM cells only; (-E) the cells were pretreated at 37 C for 4 h with genistein at varied concentrations (, 5,, μg/ml, respectively), and then incubated with probe (5 μm). Scale bar, μm. (F, F) Relative pixel intensity measurements (n = 3) from the above corresponding fluorescence images by using software ImageJ (the pixel intensities from images e and b are defined as.). (G, G) The changes of DPPIV and FP in the above corresponding cells determined by western blot (GPDH as a protein standard;.s. o significance, * p<.5, p<., as compared with the control, twosided Student s t-test). 45 C C 4 ID ( 3 ) 3 5 ID ( 3 ) 6 8 Figure S6. Representative IHC images of H89PM cells. H89PM cells were immunolabeled with () anti-dppiv antibody (control); H89PM cells were preincubated with genistein ( μg/ml) for 4 h, and then immunolabeled with () anti-dppiv antibody. H89PM cells were immunolabeled with () anti-fp antibody (control); H89PM cells were pre-incubated with genistein ( μg/ml) for 4 h, and then immunolabeled with () anti-fp antibody. Scale bar, μm. (C, C) The integral optical density (ID) values from the above corresponding IHC images; the ID values represent the relative concentrations of intracellular DPPIV and FP, respectively ( p<., two-sided Student s t-test). S-

11 C D E C D E F C D E DPPIV/GPDH G DPPIV GPDH * C D E F.S. C D E FP/GPDH G FP GPDH.S. ** C D E Figure S7. Fluorescence images of MK8 cells. (,) MK8 only. (-E) The MK8 cells were pretreated with, 5,, and μg/ml of genistein, respectively, at 37 C for 4 h, and then incubated with probe ( μm). (-E) The MK8 cells were pretreated with, 5,, and μg/ml of genistein, respectively, at 37 C for 4 h, and then incubated with probe (5 μm). Scale bar μm. (F, F) Relative pixel intensity measurements (n = 3) from the above corresponding images by using the software ImageJ (the pixel intensities from images E and are defined as.). (G, G) The changes of DPPIV and FP in the above corresponding cells determined by western blot (GPDH as a protein standard;.s. o significance, * p<.5, ** p<., p<., as compared with the control, two-sided Student s t-test). S-

12 G ** C D E F G C D E F Figure S8. Fluorescence images of MGC83 cells. () MGC83 only. (-E) The MGC83 cells were pretreated with μg/ml genistein at 37 C for, 6, and 4 h, respectively, and then incubated with probe ( μm). (F) MGC83 cells were pretreated with μg/ml genistein for 4 h, then treated with nm linagliptin for h, and finally incubated with probe ( μm). () MGC83 cells only. (-E) MGC83 cells were pretreated with μg/ml genistein at 37 C for, 6, and 4 h, respectively, and then incubated with probe (5 μm). (F) MGC83 cells were pretreated with 5 μm PT- for h, and finally incubated with probe (5 μm). Scale bar μm. (G, G) Relative pixel intensity measurements (n = 3) from above corresponding images by using the software ImageJ (the pixel intensities from images E and are defined as.; ** p<., p<., as compared with the control, two-sided Student s t-test). C D E F C D E F G ** C D E F G C D E F Figure S9. Fluorescence images of H89PM cells. () H89PM only. (-E) The H89PM cells were pretreated with μg/ml genistein at 37 C for, 6, and 4 h, S-

13 respectively, and then incubated with probe ( μm). (F) cells were pretreated with μg/ml genistein for 4 h, then treated with nm linagliptin for h, and finally incubated with probe ( μm). () H89PM cells only. (-E) H89PM cells were pretreated with μg/ml genistein at 37 C for, 6, and 4 h, respectively, and then incubated with probe (5 μm). (F) H89PM cells were pretreated with 5 μm PT- for h, and then incubated with probe (5 μm). Scale bar μm. (G, G) Relative pixel intensity measurements (n = 3) from above corresponding images by using the software ImageJ (the pixel intensities from images E and are defined as.; ** p<., p<., as compared with the control, two-sided Student s t-test) Genistein (mg/l) Genistein (mg/l) Figure S. Effects of genistein at varied concentrations on the fluorescence of reaction systems. () Effects of genistein at varied concentrations on the fluorescence of () probe (5 μm) and () its reaction product with FP (.6 μg/ml). The reactions were performed in PS (ph 7.4) at 37 C for 3 h. () Effects of genistein at varied concentrations on the fluorescence of probe ( μm) and () its reaction product with DPPIV (. μg/ml). The reactions were performed in PS (ph 7.4) at 37 C for 3 min. λ ex/em = 585/65 nm. s seen, genistein hardly affects the fluorescence of probes and and their reaction products as well as the activity of the two enzymes. C D E DPPIV FP C D C D Cell Viability (%) F Time (day) Figure S. The relationship between enzymes concentrations and cell viabilities. (, ) Fluorescence images of SGC79 and MGC83 cells incubated with probe ( μm), S-3

14 respectively. (C, D) Fluorescence images of MGC83 and SGC79 cells incubated with probe (5 μm), respectively. Scale bar μm. (E) Relative pixel intensity measurements (n = 3) from the above corresponding images by using the software ImageJ (the pixel intensities from images a and c are defined as.). The insets show the western blot analysis of the corresponding cells. (F) The viabilities of () SGC79 and () MGC83 cells (the highest cell viability is defined as %). p<., two-sided Student s t-test. DPPIV/GPDH DPPIV GPDH ** ** FP/GPDH FP GPDH * Group Group Figure S. Western blot analysis of normal and sir-transfected MGC83 cells. () DPPIV and () FP in normal (control) and different sir-transfected MGC83 cells. ote that in both panels group is the control, and groups -5 are the cells transfected with different sirs (sequences I-IV for DPPIV, and sequences I -IV for FP, respectively). s seen, group 5 in panel a and group in panel b can be chosen for the cells with the inhibition of DPPIV and FP, respectively (* p<.5, ** p<., p<., two-sided Student s t- test). S-4