Download Supplementary Information CLAVATA3 dodecapeptide modified

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.