Fig. 4: Amber suppression-mediated acetylation at K136 of TDP-43 prevents CFTR splicing, triggers the formation of nuclear droplets via its C-terminal domain. | Nature Communications

Fig. 4: Amber suppression-mediated acetylation at K136 of TDP-43 prevents CFTR splicing, triggers the formation of nuclear droplets via its C-terminal domain.

From: Sirtuin-1 sensitive lysine-136 acetylation drives phase separation and pathological aggregation of TDP-43

Fig. 4

Acetylation at K84 reduces nuclear import. a HEK293E cells cotransfected with a plasmid containing AcRS and 4xtRNATAG (E451) and with TDP-43 with an amber stop codon at different positions and a C-terminal 6xHis tag. Media was changed to 5 mM acK-containing media 24 h before lysis. Protein lysates were analyzed via Western blot. N = 3 biological replicates. Source data are provided as a Source Data file. b Cells were cotransfected with the E451 plasmid and different C-terminally 6xHis-tagged TDP-43 constructs containing amber suppression codons. Cells were fixed after 24 h in the presence of 5 mM acK. Scale bar represents 10 µm. N = 3 biological replicates. c HEK293E shTDP-43 cells cotransfected with E451, CFTR minigene, and an empty plasmid, wt or K136TAG TDP-43. After 24 h in the presence of 5 mM acK, RNA was extracted and the splicing of CFTR exon 9 was assessed via rtPCR and visualized in an agarose gel. N = 3 biological replicates. Source data are provided as a Source Data file. d HEK293E shTDP-43 cells were cotransfected with E451 plasmid and different C-terminally tagged TDP-43 constructs. Lanes 3–5 have an amber stop codon at position 136 in addition to a mutated NLS, an amber stop codon at position 84 or a deletion of the C-terminus. Cells were treated with 5 mM acK for 24 h before lysis. N = 3 biological replicates. e HEK293E shTDP-43 cells were cotransfected with E451 and different C-terminally tagged TDP-43 constructs. Cells were fixed and immunolabeled after 24 h in the presence of 5 mM acK. Scale bar represents 10 µm. N = 3 biological replicates. f Quantification of percentage of cells with 6xHis-TDP-43 protein distribution diffuse nuclear (blue bars), nuclear clumpy (grey bars), nuclear + cytosolic, diffuse (orange bars) and nuclear + cytosolic, clumpy (yellow bars). A total of 250 cells per group cotransfected with E451 plasmid and different 6xHis C-terminally tagged TDP-43 constructs. Source data are provided as a Source Data file.

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