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. 2022 Apr 9;11(8):1283. doi: 10.3390/cells11081283

Figure 3.

Figure 3

NAD+ exposure inhibits the morphological development of neurons. (A) Representative images of Tuj1 immunostaining with Ctrl and NAD+-treated aNSPCs. Scale bar, 50 μm. (B,C) The quantification results show that the NAD+ exposure significantly decreased the dendritic length (B) and the intersection number (C) of newborn neurons derived from aNSPCs. Data are presented as the mean ± SEM. Unpaired t-test. n = 30 neurons were analyzed in each group. (D) Sholl analysis results show that NAD+ exposure reduced the dendritic complexity of aNSPCs-derived neurons compared to Ctrl group. Data are presented as the mean ± SEM. Unpaired t-test. n = 30 neurons were analyzed in each group. (E) Representative images of MAP2 immunostaining with hippocampal neurons treated with NAD+. The cultured neurons were transfected with the GFP plasmid at 4 days in vitro (DIV 4). At DIV 5, NAD+ was supplemented at the final concentration of 1 mM and 2 mM. Forty-eight hours later, MAP2 immunostaining and morphological assay on the cells were performed. Scale bar, 50 μm. (F,G) The quantification results show that NAD+ exposure reduced the dendritic complexity of hippocampal neurons compared to the control group. Data are presented as the mean ± SEM. Unpaired t-test. n = 30 neurons were analyzed in each group. (H) Sholl analysis results showed that the NAD+ exposure reduced the dendritic complexity of hippocampal neurons compared to control group. Data are presented as the mean ± SEM. Unpaired t-test. n = 30 neurons were analyzed in each group. (I) Representative images of MAP2 immunostaining with hippocampal neurons treated with NAD+. The cultured neurons were transfected with GFP plasmid at 14 days in vitro (DIV 14). At DIV 15, NAD+ was supplemented at the final concentration of 1 mM and 2 mM. Cells were performed MAP2 immunostaining and morphological assay at DIV 17. Scale bar, 50 μm. (J,K) The quantification results show that NAD+ exposure reduced the intersection numbers (J) and dendritic length (K) of hippocampal neurons compared to control group. Data are presented as the mean ± SEM. Unpaired t-test. n = 30 neurons for Ctrl and 1 mM groups and n = 25 neurons for 2 mM group were analyzed. (L) Sholl analysis results show that NAD+ exposure reduced the dendritic complexity of hippocampal neurons compared to control group. Data are presented as the mean ± SEM. Unpaired t-test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.