Probing for chemotherapy-induced peripheral neuropathy in live dorsal root ganglion neurons with atomic force microscopy  Ngan Pan Bennett Au, BS, Yuqiang Fang, MS, Ning Xi, DSc, King Wai Chiu Lai, PhD, Chi Him Eddie Ma, DPhil  Nanomedicine: Nanotechnology, Biology and Medicine  Volume 10, Issue 6, Pages 1323-1333 (August 2014) DOI: 10.1016/j.nano.2014.03.002 Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Vincristine dose-dependently reduces neurite growth of DRG neurons. Fluorescence micrographs of whole DRG explants following immunostaining with anti-β-tubulin III, which highlights neurons and their neurites. (A) Untreated DRG explants exhibited longer and more extensive neurites than those explants treated with (B) 2.5 ng/ml, (C) 10 ng/ml, and (D) 50 ng/ml vincristine. Scale bar: 500 μm. (E) Total neurites of DRG explants were significantly reduced in all vincristine-treated groups. (F) The longest neurite length from DRG explants was measured. Vincristine inhibited not only the total length of neurite outgrowth but also the longest neurite length in DRG explants at all tested concentrations. (mean±SEM of triplicates; **P<0.01, one-way ANOVA, followed by Newman-Keuls post hoc test in (E) and (F).) Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 Cell surface topography analysis of (A-C) untreated and (D-F) vincristine-treated DRG neurons (10 ng/ml). Representative three-dimensional AFM height images of (A) untreated and (D) vincristine-treated DRG neurons. AFM peak force error images of (B) untreated and (E) vincristine-treated DRG neurons. Scale bar: 10 μm. High-magnification AFM height images of (C) untreated and (F) vincristine-treated DRG neurons. Scale bar: 2 μm. (G) Comparison of surface roughness of untreated and vincristine-treated DRG neurons. (mean±SEM of triplicates; **P<0.01, Student’s t-test.) Ra represents the average surface roughness. Rq represents the root-mean-squared roughness. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 Representative AFM deflection-displacement curves of (A) untreated and (D) vincristine-treated DRG neurons. Representative AFM force-indentation curves of (B) untreated and (E) vincristine-treated DRG neurons. The curve fits are based on the modified Hertz model to the data obtained from force-indentation curves (red lines in (B) and (E)). Histogram of the Young’s modulus distribution for (C) untreated and (F) vincristine-treated DRG neurons. Average Young’s modulus value of DRG neurons was reduced to 7 kPa after addition of 10 ng/ml of vincristine. The indentation was conducted at the center of cell with an indentation depth of approximately 500 nm. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 Average Young’s modulus values obtained from AFM force measurements of untreated and vincristine-treated DRG neurons at 2.5 ng/ml (low), 10 ng/ml (medium) and 50 ng/ml (high). Average Young’s modulus value was decreased with increasing concentrations of vincristine (mean±SEM of triplicates; **P<0.01, one-way ANOVA, followed by Newman-Keuls post hoc test.) Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 Confocal laser scanning microscopy revealed depolymerization of cytoskeletal microtubules in DRG neurons. (A) Well-organized microtubular scaffold was observed in untreated dissociated adult DRG neurons after immunostaining with neuronal specific marker anti-β-tubulin III. (B-D) The anti-β-tubulin III staining and the network of cellular scaffold are gradually lost in DRG neurons with increasing concentration of vincristine, as indicated by white arrows. The cytoskeleton significantly depolymerized at high concentration of vincristine (50 ng/ml). Scale bar: 10 μm. (E) Quantification of β-tubulin III fluorescence intensity after treatment with various concentrations of vincristine. Values represent mean±SEM of triplicates; **P<0.01, one-way ANOVA, followed by Newman-Keuls post hoc test. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Representative AFM deflection-displacement curves of (A) untreated and (D) paclitaxel-treated DRG neurons. Representative AFM force-indentation curves of (B) untreated and (E) paclitaxel-treated DRG neurons. Curve fits based on the modified Hertz model to the data obtained from force-indentation curves (red lines in (B) and (E)). Histogram of the Young’s modulus distribution for (C) untreated and (F) paclitaxel-treated DRG neurons. Average Young’s modulus value of DRG neurons was increased to 18 kPa after addition of 500 ng/ml paclitaxel. The indentation was conducted at the center of cell, with an indentation depth of approximately 500 nm. (G) Average Young’s modulus values obtained from AFM force measurements of untreated and paclitaxel-treated DRG neurons at 500 ng/ml. Values represent mean±SEM of triplicates; **P<0.01, Student’s t-test. Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions

Nanomedicine: Nanotechnology, Biology and Medicine 2014 10, 1323-1333DOI: (10.1016/j.nano.2014.03.002) Copyright © 2014 Elsevier Inc. Terms and Conditions