Fig. 4: Decreased expression of Phe-rich proteins in differentiated FARSA knockdown motor neuron-like NSC-34 cells and post-mortem cerebellum tissue of C9orf72 patients.
From: Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA
Western blot analysis revealed decreased expression of GOLT1B, PXMP2 and ALG10B Phe-rich proteins but not Phe-low proteins GAPDH and LAMB1 in (a) differentiated motor neuron-like NSC-34 cells. Expression levels relative to shScramble average (100%) are as follows: TSPAN5 (84.2 ± 9.8%, p-value = 0.198), PXMP2 (83.5 ± 5.0%, p-value = 0.044), GOLT1B (70.0 ± 8.5%, p-value = 0.017), ALG10B (74.4 ± 1.3%, p-value = 0.0023), GAPDH (109.6 ± 11.3%, p-value = 0.923) and LAMINB1 (125.3 ± 21.2%, p-value = 0.596). Statistics was done using nested t-test on 3 independent experiments. Expression levels of TSPAN5 and ALG10B were significantly reduced in (b) RIPA lysates extracted from frozen cerebellar gray matter of ALS/FTD cases with a C9orf72 mutation (C9 + ALS/FTD). Expression levels relative to controls (ALS/FTD cases without C9orf72 mutation (C9- ALS/FTD)) average (100%) were as follows: ALG10B (53.8 ± 10.8%, p-value (two-sided) = 0.048), TSPAN5 (60.3 ± 5.1%, p-value (two-sided) = 0.0009), GAPDH (76.7 ± 4.0%, p-value (two-sided) = 0.0004), LAMINB1 (94.1 ± 9.6%, p-value (two-sided) = 0.773). Statistics was done using unpaired Student’s t-test on twelve C9 + ALS/FTD and 12 C9- ALS/FTD biologically independent samples Stain free images represent loading comparison for all proteins above the stain free image and are vertically compressed for design reasons. Molecular weight is marked on western blot pictures in kDa. Graphs present mean values ± s.e.m. with statistical significance labeled as *p < 0.05, **p < 0.01, ***p < 0.001. c Proteomic analysis indicates that the ratio of downregulated to upregulated genes, presented with the red line, increases with the increasing % of Phe in the proteins in C9orf72-patient derived lymphoblastoid cell lines. Mass spectrometry analysis was performed on six C9orf72 patient-derived and six control biologically independent samples for each sample once. d Schematic representation (created with Biorender.com) of results. Source data are provided as a Source data file.
