1. Volume 1, Issue 2, Pages 246-263 (August 2016)
Enzyme-Instructed Self-Assembly for Spatiotemporal Profiling of the Activities of Alkaline Phosphatases on Live Cells 
Jie Zhou, Xuewen Du, Cristina Berciu, Hongjian He, Junfeng Shi, Daniela Nicastro, Bing Xu 
Chem 
Volume 1, Issue 2, Pages (August 2016)
DOI: /j.chempr
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2. Chem 2016 1, DOI: ( /j.chempr )
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3. Figure 1
EISA Forms Fluorescent, Non-diffusive Nanofibrils with High Spatiotemporal Resolution
(A) Illustration of the concept of EISA for generating fluorescent nanofibrils.
(B) Molecular structures of pNDP1 and NDP1 and the conversion catalyzed by ALP under physiological conditions (orange part is the fluorophore, NBD; red part is the enzymatic trigger, phosphate).
(C) Illustration of the EISA of NDP1 to form fluorescent pericellular nanofibrils in co-culture.
(D) Fluorescent nanofibrils of NDP1 selectively form on HeLa cells (the cancer cell, white arrows) and in HS-5 cells (the stromal cell, white arrowheads) in the co-culture of HeLa and HS-5 cells ([pNDP1] = 500 μM; 0 hr [left], 6 hr [middle], and 24 hr [right] incubation). The scale bar represents 10 μm. Nuclei were stained with Hoechst
Chem 2016 1, DOI: ( /j.chempr )
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4. Figure 2
ALP Catalyzed Molecular Self-Assembly to Form Fluorescent Nanofibrils
(A and B) TEM images of pNDP1 (A) before and (B) after treatment with ALP (2 U/mL) at a concentration of 1.0 wt % and pH 7.4. Inset: optical images of the solution and hydrogel, respectively. The scale bar represents 100 nm.
(C) Rheological characterization of the hydrogelation process by treating the solution of pNDP1 with ALP (2 U/mL) at a concentration of 1.0 wt % and pH 7.4. The gelation point (the storage modulus [G′] dominates over the loss modulus [G″]) appears at 12 min after the addition of ALP.
(D–F) Enzyme-catalyzed self-assembly of NDP1 at 20 μM (below its minimum gelation concentration). (D) SLS signals of pNDP1 without and with the addition of ALP (2 U/mL) to the solution of pNDP1 (20 μM, pH 7.4). (E) The confocal fluorescent microscope image shows the appearance of bright spots in the solution of pNDP1 (20 μM, pH 7.4) after treatment with ALP (20 U/mL). The scale bar represents 100 μM. (F) TEM image of NDP1 formed after treatment of pNDP1 (20 μM, pH 7.4) with ALP (2 U/mL). Scale bars represent 100 (main) and 50 nm (inset).
Chem 2016 1, DOI: ( /j.chempr )
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5. Figure 3
CLEM Confirms Pericellular Fluorescent Nanofibrils on HeLa Cells
(A) Merged image (differential interference contrast and fluorescence microscopy images) of treated HeLa cells (grown on marked Aclar discs and incubated for 6 hr with 500 μM pNDP1), recorded only a few minutes before the sample was high-pressure frozen for TEM; note the highest intensity of fluorescence signal on the cell surface, indicating a high abundance of nanofibrils in that region of the cell.
(B) High-magnification electron microscopy image shows a slice through the same cell dipicted in (A). Note that this is a montage image that consists of more than 200 individual high-magnification image tiles.
(C) Higher-magnification electron micrograph of the cell region highlighted by the red box in (A) and (B). This region of the cell also corresponds to the area of highest fluorescence signal in (A).
(D) High-magnification electron micrographs of the red boxed area in (C). Note the presence of nanofibrils (white arrowheads), containing NDP1s.
(E) 3D-rendered graphical model of the 3D tomographic reconstruction (∼70-nm-thick plastic section) shown in (D).
Scale bars represent 2,000 nm (A and B) and 200 nm (C and D).
(F) High-magnification electron micrograph of a whole control cell (wild-type, untreated cell). Note that this is a montage image that was stitched together from more than 200 individual high-magnification image tiles.
(G) Higher-magnification electron micrograph of the region highlighted by a blue box in (F).
(H) High-magnification electron micrographs of the blue boxed area in (G). Note the absence of nanofibrils. Scale bars represent 2,000 nm (F), 500 nm (G), and 200 nm (H).
Chem 2016 1, DOI: ( /j.chempr )
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6. Figure 4 EISA for Profiling the Activities of ALP on Live Cells
(A–E) Confocal microscope images show (A) the time course of fluorescence on the HeLa cells incubated with pNDP1 for 30 min, 1 hr, 3 hr, and 6 hr (blue pseudocolor, Hoechst 33342), (B) 3D stacked z-scan images of fluorescence on HeLa cells treated with pNDP1 for 6 hr, (C) no fluorescence on HeLa cells after the cells from (A, 6 hr) were re-incubated in fresh medium for 36 hr, (D) fluorescence emission on two pairs of drug-sensitive and drug-resistant cancer cell lines (A2780 and A2780cis; MES-SA and MES-SA/Dx5, 24 hr) incubated with pNDP1, and (E) fluorescence on cancer cells (MCF-7) incubated with pNDP1 without (upper) and with (bottom) the addition of prednisolone. Scale bars represent 20 μm (A, upper row, and C) and 5 μm (A, lower row, D, and E).
(F) The amount of ALP on HeLa cells according to the number of pericellular nanofibrils was measured by the fluorescence intensity of the molecules remaining on the cell surface.
(G) Time is plotted against the fluorescence of pericellular nanofibrils on HeLa cells treated with pNDP1. The initial cell number was 5,000 cells/well.
(H) Confocal images (scale bar represents 10 μm) of different cell lines incubated with pNDP1. The incubation time was 24 hr for HS-5, PC3, U-87 MG, Capan-2, A375, SKOV3, PC-12 Adh, and T98G and 12 hr for Saos-2 and 7F2. The incubation concentration of pNDP1 was 100 μM for Saos-2 and 7F2 and 500 μM for other cells. All the confocal images were taken after removal of the medium with pNDP1 and then the addition of live-cell image solution.
Chem 2016 1, DOI: ( /j.chempr )
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