The effect of fluorescent nanodiamonds on neuronal survival and morphogenesis

Yung-An Huang1,2,#, Chun-Wei Kao1,3,#, Kuang-Kai Liu1, Hou-Syun Huang1,4,Ming-Han Chiang3, Ching-Ren Soo3,Huan-Cheng Chang4, Tzai-Wen Chiu1,3,*, Jui-I Chao1,3,*, and Eric Hwang1,2,3,*

1Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan

2Institute of Bioinformatics and Systems Biology, National Chiao Tung University, Hsinchu30068, Taiwan 30068

3Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan

4Institute of Atomic and Molecular Sciences, Academia Sinica, Taipei10672, Taiwan

Figure S1. FNDs did not induce the activation of caspase-3 in dissociated hippocampal neurons. Quantification of the level of activated caspase-3 in hippocampal neurons treated with various dosages of FNDs. 10 μM of sodium arsenite (NaAsO2) was added to the culture medium 48 hours (a condition known to induce apoptosis in dissociated neurons1) before fixation. No statistically significant difference between control group (0 μg/mL FNDs) and FND-treated groups were detected (one-way ANOVA followed by Dunnett’s post-hoc test). The only statistically significant difference observed was between the control group (0 μg/mL FNDs) and neurons treated with 10 μM NaAsO2. The bar graph is expressed as mean ± SEM (N=3).

Figure S2. The particle size and morphology of FNDs under scanning electron microscope. FND particles were observed by a scanning electron microscope.

Figure S3. The size distribution of FNDs used in this study. The average size of FNDs was 114.7 nm as determined by dynamic light scattering.

Figure S4. Schematic diagram of the intracranial injection and novel object recognition task.(A) The novel object recognition test (NORT) was assessed before the intracranial injection at day 8. FNDs or saline were intracranially injected into rat hippocampi using the stereotaxic instrument. After the surgery, the body weight, fodder and water consumption were monitored on a daily basis. NORT was assessed again 8 days after the surgery (day 16). (B) NORT consisted of the training and testing sessions and were separated by a retention interval of 1 hour. During the training session, the rat was placed in the box at the center of two identical objects and allowed to explore for 10 mins. After the 1 hour retention, a novel object replaced one of the familiar objects. During the testing session, the rat was return to the box and exposed to the objects for 10 mins.

Reference:

1.Namgung, U. & Xia, Z. Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase. J. Neurosci.20, 6442-6451 (2000).