Skip to main content
. 2020 Apr 13;23(5):101058. doi: 10.1016/j.isci.2020.101058

Figure 4.

Figure 4

Tightly Regulated Nwd1 Expression Is Required for the Induction of Neuronal Identity

(A–G) A non-targeting shRNA (A–C) or Nwd1 shRNAs (D–F) were electroporated together with EGFP into primary cultured cortical neurons. Neurofilaments were stained with an anti-SMI312 antibody (red) at 3 div. Nuclei were stained with Hoechst dye (blue). (G) Number of SMI312+ axons extending from a single neuron. ∗∗p < 0.01 (chi-square test); control shRNA, n = 101; shRNA #1, n = 108; shRNA #2, n = 99.

(H–N) Cortical neurons transfected with EGFP-Nwd1 (K–M) or control EGFP (H–J) were stained for SMI312 (red) at 3 div. Nuclei (blue) (N) Number of SMI312+ axons extending from a single neuron. ∗∗∗p < 0.001 (chi-square test); EGFP, n = 149; EGFP-Nwd1, n = 149.

(O–U) EGFP-Nwd1 or control EGFP were electroporated into cortical neurons and cultured for 1 div (O and R), 2 div (P, S), and 3 div (Q and T). To visualize fine immature neurites, a dsRed expression plasmid was co-electroporated into the cells. (U) Number of neurites extending from a single neuron. Data are presented as means ± SEM. ∗∗∗p < 0.01, Welch's t test; EGFP 1 div, n = 150; 2 div, n = 150; 3 div, n = 200; EGFP-Nwd1 1 div, n = 150; 2 div, n = 150; 3 div, n = 200. Scale bars, 20 μm.