Fig. 4

C21ORF2-V58L causes apoptosis, reduced mitochondrial activity and impaired DNA damage responses. A–L Expression and functional analysis of iPSC-derived motor neuron (MN) cultures at days in vitro 25 (DIV25, 7 days after plating). C1 and C2, healthy controls; P1, ALS patient with C21ORF2-V58L mutation; P2, ALS patient with C21ORF2-V58L/C61Y mutations. A–E C21ORF2 protein and mRNA expression is unchanged in mutant MNs A, B Co-immunostaining for CHAT and C21ORF2 in control and mutant. B Quantification of immunostainings as in A. C RT-qPCR analysis of C21ORF2 gene expression in control and mutant MNs. For relative mRNA expression the ΔCt values (ΔCt = Ct(C21ORF2) – Ct (ACTINB)) for each line are shown. D, E Western blot analysis using anti-C21ORF2 and anti-β-ACTIN antibodies. β-ACTIN was used as loading control. D Representative Western blot. E Quantification of C21ORF2/actin intensity ratio in Western blot experiments as in D. F, G The number of cCas3-positive MNs is increased in mutant cultures. F Co-immunostaining for cleaved-caspase-3 (cCas3) and CHAT in control and mutant cultures. G Quantification experiments as in F. H Representative kinetics graphs of mitochondrial oxygen consumption in cultures. I, J Quantification of maximum respiration and spare respiratory capacity, both of which show a significant reduction in mutant cultures. K, L Co-immunostaining for CHAT and yH2AX, a marker of the DNA damage response, after 1 h of neocarzinostatin (NCS) treatment. L Quantification of yH2AX staining intensity in MN nuclei after NCS treatment, showing a significant decrease in mutant MNs. B–E, G, I, J, L Results of at least three independent experiments are plotted as means with SEM. *p < 0.05; ** p < 0.01; ****p < 0.0001, G, I, J, L Kruskal-Wallis test, Dunn’s multiple comparison post-hoc test. Scale bars: A, 10 µm; F, 100 µm; K, 5 µm