Fig. 3: Expression of Phe-containing proteins is decreased in HEK293T cells, FARSA knockdown HEK293 cells and C9orf72 patient-derived lymphoblastoid cell lines.
From: Phenylalanine-tRNA aminoacylation is compromised by ALS/FTD-associated C9orf72 C4G2 repeat RNA
a Click chemistry experiment in HEK293T cell line stably expressing mutated FARSA protein showed reduced global expression levels of Phe-containing proteins in the presence of 32×C4G2 RNA. Phe-content normalized to whole protein expression and relative to S1m transfected cells was for RFP 105.1 ± 6.83 and for 32×C4G2 81.4 ± 5.64 (p-value (two-sided) = 0.037). Statistics was done using unpaired Student’s t-test on four independent experiments. The Western blot analysis of Phe-rich protein expression levels in (b) FARSA knockdown HEK293 cells and (c) C9orf72 patient-derived lymphoblastoid cells showed decreased expression of Phe-rich proteins TSPAN5, PXMP2, GOLT1B, and ALG10B whereas there was no decrease in expression of Phe-low proteins LAMINB1 and GAPDH. Expression levels in FARSA knockdown HEK293 cells relative to shScramble average (100%) are as follows: TSPAN5 (76.2 ± 10.6%; p-value = 0.023), PXMP2 (81.1 ± 10.9%; p-value = 0.026), GOLT1B (87.5 ± 3.1%; p-value = 0.048), ALG10B (77.9 ± 9.4%; p-value = 0.049), GAPDH (101.6 ± 6.5%; p-value = 0.897) and LAMINB1 (106.1 ± 7.1%; p-value = 0.249). Statistics was done using nested t-test on 4 independent experiments. Expression levels in lymphoblastoid cell lines are as follows: 14.41 ± 4.46 in control and 8.63 ± 3.55 in C9orf72 patient-derived cells for TSPAN5 (p-value (two-sided) = 0.033); 10.1 ± 2.15 in control and 3.88 ± 1.68 in C9orf72 patient-derived cells for PXMP2 (p-value (two-sided) = 0.00028); 15.75 ± 3.6 in control and 11.22 ± 1.88 in C9orf72 patient-derived cells for GOLT1B (p-value (two-sided) = 0.0027); 9.39 ± 0.27 in control and 8.44 ± 0.24 in C9orf72 patient-derived cells for ALG10B (p-value (two-sided) = 0.038); 9.350 ± 0.508 in control and 10.036 ± 0.507 in C9orf72 patient-derived cells for LAMINB1 (p-value (two-sided) = 0.362); 15.167 ± 0.586 in control and 14.430 ± 0.714 in C9orf72 patient-derived cells for GAPDH (p-value (two-sided) = 0.444). Statistics were done using unpaired Student’s t-test on six C9orf72 patient-derived and six control 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 relative to shScramble in HEK293 or control lymphoblastoid cell lines ± s.e.m. with statistical significance labeled as *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.
