Additional file 5 of A stress-dependent TDP-43 SUMOylation program preserves neuronal function

Supplementary Material 5. Fig. S1: Characterizing stress responsive TDP-43 SUMOylation. (A) Representative immunofluorescent microscopy images of TDP-43 and G3BP1 and western blot from SUMOylation assays testing whether various stressors that cause TDP-43 aggregation induce TDP-43 SUMOylation: NaAsO2 (250 μM), D-Sorbitol (400 mM), NaCl (300 mM), Heat Shock (42 °C) in HEK293T (Immunofluorescence) and HEK293T HA-SUMO2 stable cells (biochemistry). One-Way ANOVA with Fisher’s LSD test. Data presented as mean ± SEM. *p 0.05. For all analyses wild type mice are presented in grey and K408R/K408R mice are presented in purple. Fig. S12: Characterizing the molecular and histological effects of blocking TDP-43 SUMOylation in vivo. (A) Representative western blot and quantification of TDP-43 and phosphorylated TDP-43 in the RIPA and UREA fractions of the cortex from male mice at 2, 9, and 16 months of age (2MO, 9MO, and 16MO, respectively). Data presented as mean ± SEM. (N = 4 per genotype) 2-Way ANOVA with Fisher’s LSD test. (B) Representative western blot and quantification of TDP-43 and phosphorylated TDP-43 in the RIPA and UREA fractions of the cortex from male mice at 2, 9, and 16 months of age (2MO, 9MO, and 16MO, respectively). Data presented as mean ± SEM. (N = 4 per genotype) 2-Way ANOVA with Fisher’s LSD test. *p 80 NMJs were quantified per animal. Data presented as mean ± SEM. (N = 3-4 per genotype) 2-Way ANOVA with Tukey’s multiple comparison analysis. (H) Representative images and quantification of ChAT+ motor neurons in the ventral horn of the lumbar spinal cord of male and female mice. Each datapoint is the average of 4 serial sections spaced 40 μm apart through the lumbar enlargement of the lumbar spinal cord. Data presented as mean ± SEM. Unpaired t-test, ** p <0.005. For all analyses wild type mice are presented in grey and K408R/K408R mice are presented in purple. Fig. S13: Optimization of proximity ligation assay in human tissue. (A) Quantification of neuropathology score of PPFE prefrontal cortex (PFC) tissue sections (5um) from patients diagnosed with ALS/FTD and unaffected controls. Data presented as mean ± SEM. (N = 6-8) Unpaired t-test, ** p <0.005. (B) Representative images of proximity ligation assay (PLA) between TDP-43 and SUMO2/3 in human PPFE hippocampus tissue sections (5 µm). Yellow arrows indicate TDP-43:SUMO2/3 interactions where the signal specific to the PLA channel and not identified in the autofluorescence channel. Scale bar = 18 μm.