A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response

A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response

Journal Pre-proof A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response Xue Zhang, Caiy...

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Journal Pre-proof A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response Xue Zhang, Caiyun Liu, Xinyu Cai, Bin Tian, Hanchuang Zhu, Yanan Chen, Wenlong Sheng, Pan Jia, Zilu Li, Yamin Yu, Shengyun Huang, Baocun Zhu

PII:

S0925-4005(20)30167-2

DOI:

https://doi.org/10.1016/j.snb.2020.127820

Reference:

SNB 127820

To appear in:

Sensors and Actuators: B. Chemical

Received Date:

2 November 2019

Revised Date:

16 January 2020

Accepted Date:

3 February 2020

Please cite this article as: Zhang X, Liu C, Cai X, Tian B, Zhu H, Chen Y, Sheng W, Jia P, Li Z, Yu Y, Huang S, Zhu B, A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response, Sensors and Actuators: B. Chemical (2020), doi: https://doi.org/10.1016/j.snb.2020.127820

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

A highly specific Golgi-targetable fluorescent probe for tracking cysteine generation during the Golgi stress response Xue Zhang,a Caiyun Liu,a,* Xinyu Cai,a Bin Tian,b Hanchuang Zhu,a Yanan Chen,a Wenlong Sheng,b,* Pan Jia,a Zilu Li,a Yamin Yu,a Shengyun Huang,c,* and Baocun Zhu a,* a

School of Water Conservancy and Environment, University of Jinan, Jinan 250022,

b

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China.

Biology Institute, Qilu University of Technology (Shandong Academy of Sciences),

Jinan 250103, China.

Department of oral and maxillofacial surgery, Shandong Provincial Hospital

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c

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Affiliated to Shandong University, Jinan 250021, China.

E-mail address: [email protected] (C. Liu), [email protected] (B. Zhu),

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[email protected] (W. Sheng), and [email protected] (S. Huang)

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Graphical Abstract

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Highlights 

The probe could quantitatively and sensitively detect Cys (DL=5.1×10−8 mol/L)



The probe displayed prominent selectivity to Cys than other species including biothiols The probe could specifically target the Golgi apparatus



The probe could be applied to tracking basal Cys in live cells and zebrafish



The probe could track the generation of Cys during the Golgi stress response

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in live cells and zebrafish

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Abstract

In the appropriate Golgi stress response, adaptive repair will occur by changing

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or triggering the expression of related pathways. Furthermore, it also can inhibit the

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cytotoxicity of cells in the altered redox homeostasis. Elevated biosynthesis and transport of cysteine (Cys) will be induced to restore the abnormal redox state, and

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more evidences are needed to confirm this process in the Golgi stress response. Herein, we developed a novel Golgi-targetable Cys-specific fluorescent probe

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GT-Cys for sensitively detecting Cys generation during the Golgi stress response. In probe GT-Cys, thiobenzoate moiety was chosen as recognition group of Cys, and 4-CF3-7-aminoquinoline dye was selected as fluorophore and Golgi targetable unit. Probe GT-Cys can selectively and sensitively respond to Cys. Additionally, it has good targetable properties, facilitating the study of complicated stress response of 2

Golgi apparatus. Importantly, the applications of probe GT-Cys in biological imaging showed that it is sensitive enough to basal Cys, especially to endogenous Cys during the Golgi stress response. Keywords: Fluorescent probe; cysteine; Golgi stress response; Golgi-targetable bioimaging; aminoquinoline; thiobenzoate

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1. Introduction

Organelles stress response is an essential part to meet the needs of cells,

including stress response of endoplasmic reticulum (ER), mitochondria, lysosome and

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Golgi apparatus [1]. In recent years, the mechanism of organelle stress response has

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been found in detail, especially ER stress response. In contrast, Golgi stress response has not been studied extensively [2-6]. As a major organelle in most eukaryotic cells,

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Golgi apparatus is a dynamic structure of proteins packing, transport and sorting [7].

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When the cells are stimulated, they will be associated with the elevated Golgi stress response. At the same time, they will trigger the corresponding pathways to reduce

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the burden of Golgi apparatus. However, excessive Golgi stress response may contribute to apoptosis [1]. Therefore, the detailed mechanism of Golgi stress

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response should be fully clarified. Recently, the mechanism of Golgi stress response induced by appropriate cell

stimulation to restore redox homeostasis has been further disclosed. It has been confirmed that cysteine (Cys) tends to increase during the Golgi stress response to provide cytoprotection [8]. As the precursor of glutathione (GSH), Cys can participate 3

in the antioxidant defense system together with GSH to provide protection for cells [9]. The redox state in cells is determined by the amount, production rate and consumption rate of reactive oxygen species (ROS) and reducing substances, as well as the interaction between these species [10]. When oxidative stress occurs in living cells, redox equivalence can be restored by changing the production of Cys.

important to expound its cytoprotection functions.

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Obviously, tracking the production of Cys during the Golgi stress response is very

As a non-invasive and high spatial resolution method, fluorescent probe has been widely used in the tracking of bioactive molecules in living cells and in vivo, and

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provide an effective way to explore physiological and pathological functions of these

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species [11-12]. In recent years, a large number of fluorescent probes have been developed to detect Cys by using its nucleophilic and reductive properties. The related

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mechanisms include Michael addition, cracking reaction, cyclization reaction, and so

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on [13-22]. Especially, organelle-targeted fluorescent probes are of great significance in detecting bioactive molecules in situ and dynamically. For instance, Fan’s group

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developed a ratiometric and mitochondrial-targeted fluorescent probe to visualize Cys/Hcy [23]. Ye’s group developed an ER-targeted fluorescent probe to study GSH,

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Cys and HOCl in ER stress [24]. Yin’s group developed a lysosome-targeted fluorescent probe to detect lysosomal cysteine in situ, in order to explore the relationship between apoptosis and lysosomal Cys [25]. Although many outstanding probes have been used in Cys detection, there are still some aspects to be improved. First of all, it is a severe challenge to distinguish Cys from other biothiols such as Hcy, 4

GSH and H2S by fluorescent probes. Among them, some probes distinguish Cys from Hcy according to their different reaction rates with Cys and Hcy respectively. However, in the long-term reaction between the probe and Cys, the interference caused by Hcy cannot be avoided. Secondly, despite the wide applications of fluorescent probes in specific organelles [26-32], specific Golgi-targeting probes are rarely reported [33-46]. Especially, Cys, as one of the major biothiol in biological

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system, its function in the Golgi apparatus deserves further study.

In this respect, we are committed to develop a novel fluorescent probe, which has the ability to target the Golgi apparatus, and can sensitively and selectively detect

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endogenous Cys. In order to monitor Cys specifically, probe GT-Cys was constructed

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by combining thiobenzoate with aminoquinoline derivative. The thiobenzoate moiety was selected as the recognition group because of its notable selectivity to Cys [16].

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Aminoquinoline derivative was chosen as fluorophore because of its excellent

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Golgi-targetability and convenient synthesis [41,45,46]. As expected, probe GT-Cys was successfully applied to the specific detection of Cys and has the satisfactory

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feature of targeting the Golgi apparatus. In addition, based on the lower cytotoxicity, probe GT-Cys also has been utilized to monitor basal Cys in living cells and zebrafish.

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Most importantly, probe GT-Cys can be served as a powerful tool to monitor the production of Cys under elevated Golgi stress.

2. Experimental section 2.1 General Unless otherwise stated, for fluorescence spectra measurements, the probe 5

GT-Cys was dissolved in ethanol for stock solution, and the test samples were obtained in the solution of PBS/ethanol (8:2, pH = 7.4, 10 mM PBS). Upon the addition of analytes, the fluorescence intensity of probe GT-Cys at 518 nm was recorded after 30 min at 25 °C, and the slit widths were 4 nm. Bioimaging experiments were carried out under confocal fluorescence microscope, selecting different type of cells and five-day-old zebrafish as samples. Furthermore, the

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utilization of materials and instruments were detailed in the Supporting Information. 2.2 Synthesis of probe GT-Cys

Phenyl chlorothionocarbonate (173 mg, 1.0 mmol) and compound 1 (288 mg, 1

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mmol) was dissolved in tetrahydrofuran (6 mL). The resulting solution was stirred at

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room temperature for 12 h. The reaction solvent was removed under reduced pressure. Then, the residue was purified by silica gel column chromatography to afford a white

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solid. 1H NMR (400 MHz, CDCl3) δ (ppm): 7.203(d, J = 8.0 Hz, 2H), 7.336(t, J = 7.4

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Hz, 1H), 7.464(t, J = 7.8 Hz, 2H), 7.515-7.578(m, 4H), 8.122-8.187(m, 5H), 8.910(s, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm): 115.70, 119.51, 120.79, 122.03, 122.62,

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124.76, 124.94, 126.68, 127.50, 127.63, 129.10, 129.49, 130.40, 134.97, 137.92, 149.54, 153.13, 157.68. HRMS (ESI): Calcd for C23H16F3N2OS [M+H]+ 425.0935;

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Found, 425.0932.

3. Results and discussion 3.1 Optical properties of probe GT-Cys toward Cys Thiobenzoate, as a novel Cys receptor reported by Tang et al, was introduced to 6

specifically and sensitively recognize Cys via the nucleophilic addition reaction [16]. On the other hand, 4-CF3-7-aminoquinoline as a Golgi-targeting group has been fully confirmed by several newly published studies [41,45,46]. With the benefits of enhanced intramolecular charge transfer (ICT) structure and relatively high fluorescence quantum yield, 4-CF3-7-aminoquinoline is an ideal candidate for constructing novel Golgi-targeting fluorescent probes. The possible detection

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mechanism was also further confirmed by HRMS (see the Supporting Information)

and shown in Scheme 2. The fluorescence behaviors of GT-Cys (5 μM) to Cys were

tested in the solution of PBS/ethanol (8:2, pH = 7.4, 10 mM PBS). The probe solution

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exhibited very weak fluorescence (Φ = 0.007), which is ascribe to the suppressed ICT

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process induced by C=S group and enhanced photo-induced electron transfer (PET) effect of the sulfur atom and phenyl moiety. As expected, the fluorescence intensity at

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518 nm increased significantly after adding Cys (Φ1 = 0.237 and Φ2 = 0.106), and

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achieved maximum within 25 minutes (Fig. 1). Accordingly, the fluorescence intensity was recorded within 30 minutes. In addition, the absorption spectra of probe

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GT-Cys were recorded in the presence and absence of Cys (Fig. S1). As expected, the probe GT-Cys was stable under different conditions, such as wide physiological pH

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ranges and light irradiation (Fig. S2-S4).
3.2 Sensitivity and quantification of probe GT-Cys Subsequently, the relative fluorescence intensities with different concentration 7

Cys were recorded (Fig. S5). Upon the gradual addition of Cys, the fluorescence intensities of probe GT-Cys at 518 nm enhanced linearly with the increasing concentrations of Cys (Fig. 2a). In the range of 0-40 μM, the linear equation was y = 41807 × [Cys] (μM) + 123473, and the correlation coefficient was 0.9932 (Fig. 2b). Furthermore, the fluorescence intensity enhanced obviously with the addition of Cys (~ 110-fold), and the detection limit was calculated to be 51 nM (3σ/k). It implied that

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probe GT-Cys could effectively function in monitoring low concentration Cys under physiological conditions.


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3.3 Selectivity of probe GT-Cys

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In theory, probe GT-Cys can distinguish Cys from other biologically related species. To confirm the specificity of probe GT-Cys, the fluorescence intensities of

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probe GT-Cys were detected in the presence of common biological species, including

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cations, anions, amino acids and ascorbic acid (AA). As depicted in Fig. 3a, only Cys can induce significant enhancement of fluorescence intensity. Meanwhile, other

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species not only have no obvious effect on the fluorescence of the probe GT-Cys, but also have negligible interference on the process of identifying Cys. Besides, through

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studying the fluorescence intensity of the probe GT-Cys in the presence of Cys, Hcy, GSH and H2S for a long time, it is found that the probe GT-Cys has good selectivity for Cys (Fig. 3b). Especially, Hcy, even though it has the nucleophilicity and reducibility similar to Cys, does not cause the fluorescence enhancement of the probe GT-Cys. Finally, the influence of ROS to probe GT-Cys has been tested, and no 8

obvious alteration of fluorescence intensity has been induced by ROS (Fig. S6). These competitive experiments show that the probe GT-Cys has high specificity for Cys in biological system.
3.4 Imaging of Cys in living cells Based on the above tests, the feasibility of detecting and imaging Cys in living

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cells with probe GT-Cys was evaluated. To explore the viability of HeLa cells in the

presence of GT-Cys, cell counting kit-8 (CCK-8) method was performed. The results showed that the cytotoxicity of GT-Cys at different concentrations to living cells was

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negligible (Fig. S7). Then, we tested its capability for monitoring exogenous and

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endogenous Cys in living cells. No obvious fluorescence was observed in the untreated cells, but it was observed in the probe-treated cells (Fig. 4a-b). The robust

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fluorescence changes demonstrated that GT-Cys could be used to monitor

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intracellular basal Cys. In the third group, the cells were firstly pretreated with Cys, followed by GT-Cys. As shown in Figure 4c, the remarkable fluorescence

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enhancement was obtained (Fig. 4c). Moreover, the N-ethylmaleimide (NEM) preincubated cells inhibited the production of Cys, and further incubated with probe

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GT-Cys. Compared to the probe-treated cells, the fluorescence in the image of these cells was decreased (Fig. 4d). The above results imply that probe GT-Cys can be served as a suitable candidate to detect intracellular Cys with high sensitivity and selectivity.
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3.5 Imaging of Cys in zebrafish With the notable performances of probe GT-Cys in imaging intracellular Cys, the zebrafish imaging experiments were carried out to explore the applicability of probe GT-Cys in vivo. As depicted in Figure 5a-b, the zebrafish treated with GT-Cys manifested a moderate fluorescence with the control zebrafish. In contrast, the zebrafish pretreated with Cys led to obvious fluorescence response, indicating its

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ability to specifically detect Cys in zebrafish (Fig. 5c). Finally, it was observed that the fluorescence was weakened in the NEM pretreated zebrafish (Fig. 5d). These

results taken together suggested the feasibility of probe GT-Cys in monitoring Cys in

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vivo.

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3.6 Golgi localization capability of probe GT-Cys

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The co-localization experiments were carried out to verify whether or not probe

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GT-Cys can locate at the Golgi apparatus. Its Golgi-targeting ability was investigated in HeLa cells with GT-Cys and commercial Golgi tracker (BODIPY TR Ceramide).

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Besides, the co-localization ability was also confirmed by the co-incubated experiments of probe GT-Cys with Mito-Tracker Red CMXRos, Lyso-Tracker Red

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DND-99 and ER-Tracker Red respectively. As shown in Fig. 6a, the green channel (GT-Cys) and red channel (BODIPY TR Ceramide) was overlapped very well in HeLa cells, and the intensity distribution of linear regions of probe GT-Cys and BODIPY TR Ceramide is coincident. In addition, the Pearson coefficient of probe GT-Cys and BODIPY TR Ceramide is 0.91, while those of probe GT-Cys and 10

LysoTracker Red DND-99, probe GT-Cys and MitoTracker Red CMXRos, probe GT-Cys and ER-Tracker Red was 0.57, 0.63 and 0.61, respectively (Fig. 6b-d). What’s more, the co-localization experiments were also carried out in other types of cells, such as PC12 and HEK293 cells, and the corresponding Pearson coefficient was 0.94 and 0.93 (Fig. S8). The above satisfactory results demonstrated that GT-Cys has desired Golgi targeting properties.

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3.7 The Golgi stress response

Next, probe GT-Cys was applied to explore the relationship between Cys and

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Golgi stress response. Monensin (Mone), as a Golgi stress-inducing agent, can induce

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the expression of cystathionine γ-lyase (CSE), activating the synthesis of Cys [8]. Hence, we pretreated HeLa cells with 0.5 μM, 1 μM and 2 μM Mone to induce Golgi

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stress, and observed the Cys produced in this process by probe GT-Cys. As shown in

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the Fig. 7, the fluorescence intensity of probe GT-Cys increased correspondingly after incubation of different concentrations of Mone for 18 h, indicating the production of

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Cys in response to the stimulation of Mone. Therefore, it can be concluded that probe GT-Cys was capable of tracking the changes of Cys under the process of Golgi

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apparatus regulating stress response.


Inspired by the above exciting results of monitoring the production of Cys during the Golgi stress response in living cells, we then tried to track the Cys produced in the process of Golgi stress response in vivo. Five-day-old zebrafish were treated with 11

different levels of Mone for 18 h, and then continued to incubate with probe GT-Cys for 30 min. Compared with the zebrafish loaded with only probe GT-Cys, Mone-pretreated zebrafish represented a distinct green fluorescence imaging (Fig. 8). This is probably that Cys has been produced under Golgi stress response in vivo. Therefore, it has been confirmed that probe GT-Cys might play a significant role in monitoring Cys under Golgi stress.

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4. Conclusions

In summary, we described a novel Golgi-targetable fluorescent probe GT-Cys to

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monitor the upregulation of Cys during the Golgi stress response. The probe can

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sensitively and specifically detect Cys via the specific reaction between Cys and the thiobenzoate moiety. Besides, the Golgi-targetable property of probe GT-Cys is good

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in different kinds of cells, and can distinguish other organelles from Golgi apparatus.

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Moreover, probe GT-Cys showed excellent performance of detecting basal Cys in living cells and zebrafish. Particularly, probe GT-Cys also could detect the generation

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of Cys in the adaptive repair by the Golgi stress response, and provided an effective method for promoting the further study of Golgi stress response.

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Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements We gratefully acknowledge financial support from the National Natural Science Foundation of China (21607053 and 21777053), Shandong Provincial Natural Science 12

Foundation (ZR2017MB014 and ZR2018PC013), and the project of the Shandong Province Higher Educational Science and Technology Support Program for Young Innovation (2019KJD005), and Youth Fund of Shandong Academy of Science (2019QN001).

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highly

specific

fluorescent

probe,

Chem.

Commun.

(2020)

https://doi.org/10.1039/C9CC08796F.

Author Biographies

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Xue Zhang studies in School of Water Conservancy and Environment, University of Jinan as postgraduate. Caiyun Liu is a lecturer in School of Water Conservancy and Environment, University of Jinan. Her research interests focus on the design and synthesis of novel fluorescent probes and their biological applications. Xinyu Cai studies in School of Water Conservancy and Environment, University of Jinan as undergraduate. Bin Tian studies in Biology Institute of Shandong Academy of Sciences as postgraduate. Hanchuang Zhu studies in School of Water Conservancy and Environment, University of Jinan as postgraduate. Yanan Chen studies in School of Water Conservancy and Environment, University of Jinan as undergraduate. Wenlong Sheng is a research associate of Biology Institute of Shandong Academy of Sciences. He received the Ph.D. from the Fudan University in 2016, and his research interests include the functional assessment using cell and zebrafish models, and the study of neuromodulation. Pan Jia studies in School of Water Conservancy and Environment, University of Jinan as postgraduate. Zilu Li studies in School of Water Conservancy and Environment, University of Jinan as postgraduate. Yamin Yu studies in School of Water Conservancy and Environment, University of Jinan as postgraduate. Shengyun Huang is an associate professor in department of oral and maxillofacial surgery, Shandong Provincial Hospital Affiliated to Shandong University. His research interests focus on the pathogenesis and biological detection of oral cancer with novel fluorescent probes, and surface modification of dental implants with new materials. Baocun Zhu is a professor in School of Water Conservancy and Environment, University of Jinan. His research interests focus on the design and synthesis of novel fluorescent probes and their biological applications, and functional fluorescent nanoparticles for the determination and removal of toxic metal ions.

Figure 1. Time-course of GT-Cys (5 μM) for tracking Cys (50 μM). 20

Figure 2. (a) Fluorescence spectra of 5 μM GT-Cys upon addition of Cys at varied concentrations (0-40 μM). (b) The Linear plot of the emission intensity (518 nm) to Cys (0-40 μM). Figure 3. (a) Fluorescence responses of 5 μM GT-Cys to 50 μM Cys and other various analytes (100 μM). (b) Fluorescence intensity of 5 μM GT-Cys with 50 μM Cys and 100 μM H2S, 1 mM GSH, and 100 μM Hcy.

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Figure 4. Confocal fluorescence images of HeLa cells with excitation at 405 nm and

emission at 490-560 nm. (a2) Free cells; (b2) Treated with 10 μM GT-Cys; (c2) Treated with 100 μM Cys, and then 10 μM GT-Cys; (d2) Treated with 200 μM

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NEM, and then 10 μM GT-Cys; (e) The relative intensities of cells.

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Figure 5. Confocal fluorescence images of zebrafish with excitation at 405 nm and emission at 490-560 nm. (a2) Free zebrafish; (b2) Treated with 10 μM GT-Cys;

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(c2) Treated with 100 μM Cys, and then 10 μM GT-Cys; (d2) Treated with 200

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μM NEM, and then 10 μM GT-Cys; (e) The relative intensities of zebrafish. Figure 6. Confocal images co-stained by (a1, b1, c1, d1) GT-Cys and (a2, b2, c2, d2)

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organelle markers in HeLa cells pretreated with 100 μM Cys. (1) Green channel: GT-Cys with excitation at 405 nm and emission at 490-560 nm. (2) Red channel:

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Golgi tracker with excitation at 594 nm and emission at 600-700 nm, Lyso tracker with excitation at 559 nm and emission at 580-620 nm, Mito tracker with excitation at 578 nm and emission at 590-640 nm, and ER tracker with excitation at 633 nm and emission at 650-750 nm.

Figure 7. Confocal fluorescence images of Golgi stress response in HeLa cells with 21

excitation at 405 nm and emission at 490-560 nm. (a2) the cells treated with GT-Cys (10 μM) only; (b2) the cells pretreated with Mone (0.5 μM) and then with GT-Cys (10 μM); (d2) the cells pretreated with Mone (1 μM) and then with GT-Cys (10 μM); (d2) the cells pretreated with Mone (2 μM) and then with GT-Cys (10 μM); (e) The relative intensities of cells. Figure 8. Confocal fluorescence images of Golgi stress response in zebrafish with

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excitation at 405 nm and emission at 490-560 nm. (a2) the zebrafish treated with GT-Cys (10 μM) only; (b2) the zebrafish pretreated with Mone (0.5 μM) and

then with GT-Cys (10 μM); (d2) the zebrafish pretreated with Mone (1 μM) and

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then with GT-Cys (10 μM); (d2) the zebrafish pretreated with Mone (2 μM) and

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then with GT-Cys (10 μM); (e) The relative intensities of zebrafish.

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Figure 8.

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Scheme legends Scheme 1. The synthesis of fluorescent probe GT-Cys

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Scheme 2. The response mechanism of fluorescent probe GT-Cys for Cys

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Schemes

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Scheme 1.

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Scheme 2.

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