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. 2022 Feb 14;9:820105. doi: 10.3389/fcell.2021.820105

FIGURE 2.

FIGURE 2

Other filament proteins are expressed in NEFL KO and patient iPSC-MN. (A) NEFL KO motor neurons express other neurofilament proteins. Representative immunoblot showing presence of all other tested neurofilament proteins in NELF KO and PT Day 35 motor neurons. NFL = NFL antibody (Sigma) and TUJ1 = TUBB3 antibody. (B) Quantification of A. Full-length NFL is indistinguishable from background (p < .0001) and NFH is reduced in KO and patient motor neurons (p < .0001). NFM or INA levels are not changed, and PRPH is reduced only in PT (p < .0001). n = 6-7 from two independent differentiations. Signal intensity normalized to TUBB3 protein signal. (C) RT-qPCR shows reduction (p < .0001) in NEFL mRNA expression. The residual amount of NEFL mRNA is approximately 10% of control levels. Expression levels are normalized to TUBB3. n = 6-7 from two independent differentiations. (D) All differentiated motor neurons (days 35–39) grow elaborate neurite networks positive for TUBB3 (TUJ1), NFM, INA, NFH, and PRPH. Scale bar 100 µm. (E) Quantification of NFL from immunocytochemistry shows reduction in NFL signal with C- and N-terminal antibodies (p < .0001). n = 4 from two independent differentiations. Data shown in all as mean with SD. *p < .0001. One-way ANOVA followed with Dunnett’s multiple comparison post hoc test vs. CTR. # indicates abnormal karyotype.