Fig. 5: Recruitment to PML limits the aggregation of TDP-43.
From: Induced proximity to PML protects TDP-43 from aggregation via SUMO–ubiquitin networks
a, Top, solubility assay. HEK293T cells were transfected as indicated for 48 h, exposed to HS (43 °C, 1 h) or left untreated. Where indicated, rapamycin was added (100 nM, 4 h). Bottom, unfractionated lysates. Statistical analysis is provided in Extended Data Fig. 6a (n = 3). b, Left, solubility assay as in a, but cells were treated with MG132 (25 µm, 4 h). Right, unfractionated lysates. Statistical analysis is provided in Extended Data Fig. 6b (n = 3). c, Left, solubility assay as in a and b, but cells were treated with DBeQ (20 µm, 4 h). Right, unfractionated lysates. Statistical analysis is provided in Extended Data Fig. 6c (HS, n = 5; SA, n = 4). d, Left, immunofluorescence images of pre-extracted HeLa cells transfected with FRB–PML–Myc and HA–TDP-43–FKBP. Treatment with rapamycin (100 nM, 4 h), DMSO and SA (0.5 mM, 1 h) where indicated. Scale bar, 5 µm. Right, VCP/FRB-signal ratio in PML NBs was determined per cell. At least 71 cells per condition were measured. P values of two-tailed, unpaired Student’s t tests are indicated. e, Left, solubility assay as in a–c, but cells were expressing ALS-associated TDP-43 variants as indicated. Right, unfractionated lysates. Statistical analysis is provided in Extended Data Fig. 6d (n = 3). f, Solubility assay in engineered HeLa cells expressing TDP-43–FKBP–HA from the endogenous TDP-43 locus together with FRB–PML–Myc. Treatments—rapamycin (100 nM, 4 h), HS (43 °C, 1 h) and SA (0.5 mM, 1 h). Statistical analysis is provided in Extended Data Fig. 6e (n = 3). g, Left, solubility assay as in f, but where indicated cells were treated with TAK-243 (10 µm, 4 h). Right, unfractionated lysates. Statistical analysis is provided in Extended Data Fig. 7a (n = 3). h, Fluorescence microscopy. mRuby3–PML and mClover3–TDP-43 expression from a lentiviral expression cassette (HeLa) was induced by doxycycline (1 µg ml−1, 24 h). Colocalizing and noncolocalizing foci of mRuby3–PML and mClover3–TDP-43 are indicated by yellow and white circles, respectively. Scale bar, 2 µm. Statistical analysis is provided in Extended Data Fig. 7b. i, Solubility assay as in a, but in HEK293T cells expressing FRB–Sp100–Myc (instead of FRB–PML–Myc) and HA–TDP-43–FKBP for 48 h. Statistical analysis is provided in Extended Data Fig. 8c (n = 3). Untr., untreated.
