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. 2017 Jul 25;76(9):789–799. doi: 10.1093/jnen/nlx062

FIGURE 6.

FIGURE 6

(A) Representative fluorescence images of Fig4 and EGFP in cultured murine motor neurons at day 3 postinjection of control (CTL) or Fig4 knockout (KO) CRISPR/Cas9 plasmids (25 mg/mL). Down regulation of Fig4 was achieved in > 95% of motor neurons. Fig4 KO motor neurons show large vacuoles (arrows). Scale bar: 10 µm. (B) Daily viability of motor neurons expressing EGFP and Control KO CRISPR/Cas9 (CTL) or Fig4 KO CRISPR/Cas9 (25 µg/mL). Loss of viability of Fig4 KO motor neurons was significant at day 4. *p < 0.05 versus CTL KO 1-way ANOVA, Tukey’s HSD post hoc analysis. (C) Increase in the percentage of motor neurons presenting vacuoles as shown in panel A at day 3 post injection of plasmids Control KO CRISPR/Cas9 (CTL KO) or Fig4 KO CRISPR/Cas9 (25 µg/mL). Fig4 KO toxicity in motor neurons starts at day 3. *p < 0.05 versus CTL. (D) Inhibiting TRPV4 significantly delayed loss of viability in Fig4 KO motor neurons. Daily viability of motor neurons expressing EGFP and control KO CRISPR/Cas9 (CTL) or Fig4 KO CRISPR/Cas9 (25 µg/mL) with or without treatment with the TRPV4 inhibitor GSK2193874 at 500 nM beginning post injection for how long? *p < 0.05 Fig4 KO versus CTL KO and **p < 0.05 Fig4 KO versus Fig4 KO+ GSK2193874 1-way ANOVA, Tukey’s HSD post hoc analysis.