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Supplementary Information CLAVATA3 dodecapeptide modified CdTe nanoparticles: a biocompatible quantum dot probe for in vivo labeling of plant stem cells Guanghui Yu,Yanping Tan, Xiangzhu He,Yonghua Qin, Jiangong Liang Supplementary results, figures, and references Supplementary results CdTe QDs cause BY-2 cell plasma membrane damage in long day conditions To assay the extent of QD cytotoxicity, we examined their effect on the permeability of the cell membrane. Cd2+ treatment was used as the positive control. Plasma membrane damage was assessed using the fluorescent dye SYTOX Green, a green fluorescent nuclear stain that cannot enter cells with intact plasma membranes. The number of SYTOX Green-positive cells was not obviously increased after 2 d of treatment with QDs (1.4% SYTOX Green-positive cells). However, the percentage of SYTOX Green-positive cells reached 20% with Cd2+ treatment. After 4 d of treatment, the SYTOX Green-positive cells increased slightly to 18% for QD-treated versus 40.3% for Cd2+ treatment (Figure S1). The damage caused by diffusion of CdTe QDs into BY-2 cells depends on the concentration of QDs and the treatment time (Figure S2). It is reasonable to use low CdTe concentrations and short treatments as much as possible in the bio-imaging application. Diffusion of CdTe QDs into BY-2 cells is time- and concentration-dependent To observe the behavior of QDs in BY-2 cells, we measured the extent of cell labeling over time. We found that QDs could not penetrate the BY-2 cells at either the 1.0 μM or 5.0 μM concentration at 1 d of treatment (Figure S2). However, at 2 d treatment, QDs at a concentration of 5.0 μM could penetrate the cells, but QDs at 1.0 μM still did not penetrate the cells. This suggests that the QD concentration affects uptake by cells. At the 3- and 4- d of treatment, red fluorescence was clearly observed in the cells with 3.0 μM QD treatment, and fluorescence was mainly distributed in the membranes of the cells in the 1.0 μM QD treatment (Figure S2). At 5 d of 5.0 μM QD treatment, fluorescence was observed in the nucleus. These results reflected the diffusion of QDs into the nucleus or cytoplasm with a long treatment time and most likely resulted from the toxic effects of the higher QD concentration and longer treatment. CdTe QDs also triggered intracellular ROS generation in BY-2 cells Stress causes excess production of reactive oxygen species (ROS) in the cytoplasm. The generation of excess ROS causes the modification and damage of cellular proteins, lipids, and DNA, and can lead to cell death. To evaluate whether oxidative stress was produced by QD treatment, we used the Carboxy-H2DCFDA probe. This green probe is cell permeable and, after uptake, intercellular esterase activity removes the acetate groups, allowing for oxidation by ROS to yield the fluorescent 5-(and 6)-chloromethyl-2′, 7′-dichlorofluorescein. The dye can react with H2O2 (whose formation is catalyzed by intracellular endogenous peroxidases), OH• and reactive nitrogen species. The level of green fluorescence positively correlates with the amount of ROS. In comparison with Cd2+ treatment, the fluorescence upon QD treatment was much weaker, comparable to the control (Figure S3). However, with the long incubation with CdTe QDs, cell death was obviously increased (Figure S4). Although plant cells can synthesize metal-responsive protein (PCS, phytochelatin synthesis) to chelate Cd2+ to decrease Cd2+ toxicity, long CdTe QD exposure also reduced the cells’ ability to synthesize PCS (Figure S3). Cd2+-induced more cell death than CdTe QDs Evans Blue dye can be used to detect whether a cell is alive or dead, as Evans Blue cannot travel through the intact cell membranes of healthy cells, but can penetrate the damaged cell membranes of dead cells, resulting in staining. To investigate the effect of QDs on cell death, BY-2 cells were stained with Evans Blue upon QD and Cd2+ treatment, and then imaged using light microscopy (Figure S4). The number of Evans Blue positive cells remained constant over time for the control (Figure S4A, and E). However, Cd2+ heavy metal ion stress increased the number of Evans Blue positive cells with increasing treatment time (Figure S4B, and F). The Evans Blue positive cells were not significantly increased at the 1 d treatment when cells were treated with 1.0 μM CdTe QDs (Figure S4C), but the percentage of cell death in this treatment increased with the longer treatments (Figure S4G). The change in the amount of Evans Blue positive cells for treatment with 5.0 μM CdTe QDs was demonstrated to have a similar pattern to the 1.0 μM treatment (Figure S4D, and H). The uptake of Evans Blue into BY-2 cells was also quantified (Figure S4I). These results suggest that QDs did not reduce the membrane integrity in short treatments, but long treatments decreased the viability of cells. Comparison of the effect of Cd2+ and CdTe treatment on phytochelatin synthase (PCS) expression To evaluate the toxicity of our QDs, we supplemented the culture medium of tobacco BY-2 cells with 1.0 μM QDs and 5.0 μM QDs for the entire time course, and used 1.0 μM Cd2+ as a control. The response of cells was evaluated at each day using the relative gene expression level of the phytochelatin synthesis gene, PCS. PCS is a metal-responsive protein that can chelate Cd2+ to form complexes with molecular weights of approximately 2,500 or 3,600 Da. The formation of these complexes protects the cytosol from free Cd2+ ions [1], and thus reduces the toxicity of Cd2+ taken up by cells. The results indicated that PCS showed different patterns of mRNA expression with QD or Cd2+ treatment (Figure S5). The increase in the expression level of PCS in Cd2+-stressed cells was markedly higher than in QD-treated cells at all time points. The maximum level of PCS expression was reached at the 3 d time point. However, there was no obvious difference in PCS expression between treatments with different concentrations of QDs within 1 d or 2 d (P > 0.05). These results indicate that the QDs were stable at the early time point. However, PCS expression increased at 3 d. This is perhaps due to decomposition of the QDs. PCS expression levels were lower with QD treatment than with Cd2+ treatment. These results suggest that the released Cd2+ from QDs was limited and had a definite toxic effect on BY-2 cells. Supplementary references 1. DalCorso G, Farinati S, Maistri S, Furini A (2008) How plants cope with cadmium: staking all on metabolism and gene expression. J. Integr Plant Biol 50 (10): 1268-1280.