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. 2023 Nov 13;16:1241420. doi: 10.3389/fnmol.2023.1241420

Figure 13.

Figure 13

Nitrative stress decreases retrograde transport of proNGF-KKE in aged BFCNs. Rat BFCNs were treated with either 1 mM  L-NAME or vehicle for 24 h prior to analysis of proNGF-KKE retrograde transport. ProNGF-KKE is shown in yellow, tubulin is shown in red, and DAPI is shown in blue. Yellow arrows indicate quantum dots that have accumulated in cell bodies or proximal axons. (A) ProNGF-KKE accumulated in young BFCNs treated with vehicle and aged BFCNs treated with L-NAME but not in aged BFCNs treated with vehicle. Aged BFCNs (DIV20-21) treated with 1 mM  L-NAME for 24 h exhibited increased accumulation of proNGF-KKE at the cell bodies and proximal axons [top and middle rows, quantification in (B)] relative to aged vehicle-treated controls. Aged BFCNs had greater proNGF-KKE accumulation at the axon terminals greater proNGF-KKE accumulation at the axon terminals [bottom row, quantification in (C)] relative to young (DIV8-9) vehicle-treated controls, regardless of treatment condition. Tubulin is shown in red, and proNGF-KKE is shown in yellow. (B,C) Quantification of the images shown in A. (B) Proximal axons: DIV8-9 vehicle n = 29 images, DIV20-21 vehicle n = 28 images, DIV20 L-NAME n = 30 images. The highest three points of the DIV20-21 vehicle, the highest two points of the DIV20 L-NAME, and the highest point of the DIV8-9 vehicle were detected as outliers using ROUT (Q = 1%). Removal of these outliers did not affect the significance of the comparison between DIV20 L-NAME and DIV8-9 vehicle (p = 0.2618) or between DIV20-21 vehicle and DIV8-9 vehicle (p < 0.0001). Removal of the outliers changed the comparison between DIV20-21 vehicle and DIV20 L-NAME groups from p = 0.0004 (***) to p < 0.0001 (****). (C) Axon terminals: DIV8-9 vehicle n = 29 images, DIV20-21 vehicle n = 30 images, DIV20 L-NAME n = 30 images. The highest point in the DIV8-9 group was detected as an outlier using ROUT (Q = 1%). Removal of this outlier did not affect the observed value of p. All sample sizes were generated from three chambers in three independent experiments. One-way ANOVA and post hoc Tukey test, ***p < 0.001, ****p < 0.0001. DIV, days in vitro; QD, quantum dot. Scale bars: 10 μm.