Fig. 6

Reduced protein synthesis in axons of Tdp-43 knockdown motoneurons. a Percentage of reads mapping to rRNAs from RNA-seq data of Tdp-43 knockdown (shTdp-43) and control motoneurons. Data represent mean ± SD. ***p ≤ 0.001; unpaired t-test. b Immunostaining of cultured motoneurons with antibodies against puromycin and Tau. Motoneurons were exposed to puromycin as indicated. Treatment with anisomycin was done prior to puromycin exposure. Scale bar: 20 μm. c Motoneurons were exposed to nocodazole or cycloheximide as indicated followed by puromycin labeling. Immunostaining was performed with antibodies against puromycin and Tau. Boxes indicate magnified axon segments. Scale bar: 20 μm. d Immunostaining of control and Tdp-43 knockdown motoneurons with antibodies against puromycin and Tau. Motoneurons were transduced with control or Tdp-43 knockdown lentivirus and cultured for 6 d followed by puromycin labeling. Boxes indicate magnified axon segments. Scale bar: 20 μm. e Scatter plots showing fluorescence intensity of the puromycin immunosignal in somata and axons of Tdp-43 knockdown and control motoneurons. Horizontal lines represent median. n.s. not significant, ****p ≤ 0.0001; Mann-Whitney test (N = 40 motoneurons for control and 43 motoneurons for Tdp-43 knockdown motoneurons, n = 3 independent experiments). f Scatter plot showing the axon-to-soma ratios of puromycin intensity in control and Tdp-43 knockdown motoneurons. Horizontal lines represent median. ****p ≤ 0.0001; Mann-Whitney test (N = 40 motoneurons for control and 43 motoneurons for Tdp-43 knockdown motoneurons, n = 3 independent experiments)