c elegans β tubulin levels  (Developmental Studies Hybridoma Bank)

Journal: PLOS Genetics

Article Title: Decreased Hsp90 activity protects against TDP-43 neurotoxicity in a C . elegans model of amyotrophic lateral sclerosis

doi: 10.1371/journal.pgen.1011518

Figure Lengend Snippet: A. Radial locomotion assays were used to measure motor function following temperature-based inactivation of HSP-90. Animals were shifted to the restrictive temperature at L4 stage and assessed after 24 hours of HSP-90 inactivation. Animals expressing fALS TDP-43 (M337V) combined with the hsp-90(p673) mutation show an improvement in their motility. Error bars represent Mean with 95% confidence interval (CI): N = 3 independent experimental replicates; total n>100. Statistical significance as determined using Student’s t-test. (**** p<0.0001). B. hsp-90(p673) animals did not move significantly differently from wildtype (N2) animals, which were used as non-transgenic (non-Tg) controls. N = 3 independent experimental replicates; total n>100. Statistical significance as determined using Student’s t-test. (ns = not significant). C-D. Radial locomotion assays were used to measure motor function following pharmacological inhibition of HSP-90. Treatment with the Hsp90 inhibitor 17-AAG improves motor dysfunction caused by the expression of mutant hTDP-43 in C . elegans . Two independent transgenic strains, expressing either TDP-43 (M337V) (C) or TDP-43 (A315T) (D) exhibit improved motility after 48h of 17-AAG treatment (7.5 μM) when scored at 30 minutes, but this effect diminishes or is lost by 60 minutes or 24 hours post-removal of drug. Error bars represent Mean with 95% CI. N = 4; n>100 for the TDP-43 (M337V) treated worms, and n>100 when using the TDP-43 (A315T) strain. Statistical significance as determined using a Mixed-effects analysis (Bonferroni’s multiple comparisons test post hoc test). (*** p<0.001; *p<0.05). E-F . Control worms showed no difference in their motility assessment after 48h of treatment with 17-AAG (7.5 μM). Results shown combined data from multiple experiments. Error bars represent Mean with 95% CI. N = 3; n>100. Statistical significance was determined using a Mixed-effects analysis (Bonferroni’s multiple comparisons test post hoc test). G. Radial locomotion assays were used to measure motor function in older TDP-43 (M337V) at day 3, 5, or 7 of adulthood following a 24 hour temperature-based inactivation of HSP-90. TDP-43 (M337V) ; hsp-90(p673) animals show an improvement in their motility at day 3 of adulthood, but this is lost at days 5 and 7. Error bars represent Mean with 95% CI: N = 3 independent experimental replicates; total n>65. Statistical significance as determined using Student’s t-test. (**** p<0.0001).

Article Snippet: C . elegans β-tubulin levels were measured using monoclonal antibody E7 (Developmental Studies Hybridoma Bank, 1:5000) as a loading control.

Techniques: Expressing, Mutagenesis, Transgenic Assay, Inhibition, Control

Journal: PLOS Genetics

Article Title: Decreased Hsp90 activity protects against TDP-43 neurotoxicity in a C . elegans model of amyotrophic lateral sclerosis

doi: 10.1371/journal.pgen.1011518

Figure Lengend Snippet: Hsp90 mutation or inhibition decreases accumulation of phosphorylated TDP-43 A. Representative immunoblots for total and phosphorylated TDP-43 in C . elegans samples . B-C. Mutant hsp-90 significantly decreases the levels of total and phosphorylated species of TDP-43 (pTDP-43). Error bars represent SEM: N = 4. Statistical significance as determined using unpaired t-test. (*p<0.05, **p< 0.01) D. The ratio of phosphorylated TDP-43 to total TDP-43 (pTDP-43/ total TDP-43) is unchanged in TDP43 (M337V) versus TDP43 (M337V) ; hsp-90(p673) . E. Representative immunoblots for endogenous total and phosphorylated TDP-43 in HEK293T cells pre-treated with increasing amounts of the Hsp90 inhibitor 17-AAG and exposed to Ethacrynic acid 150μM. F-G. Quantification of replicate immunoblots shows no significant differences in total TDP-43 protein levels but a trend towards a reduction in the levels of pTDP-43 in a dose-dependent manner, reaching statistical significance at the highest dose tested, 10μM. Results combined data from multiple experiments. Error bars represent SEM. N = 3. Statistical significance was determined using the One-way ANOVA test (Dunnett’s multiple comparisons post hoc test) (* p<0.05).

Article Snippet: C . elegans β-tubulin levels were measured using monoclonal antibody E7 (Developmental Studies Hybridoma Bank, 1:5000) as a loading control.

Techniques: Mutagenesis, Inhibition, Western Blot

Journal: PLOS Genetics

Article Title: Decreased Hsp90 activity protects against TDP-43 neurotoxicity in a C . elegans model of amyotrophic lateral sclerosis

doi: 10.1371/journal.pgen.1011518

Figure Lengend Snippet: A. Representative photomicrographs of proteasome reporter rpt-3p :: GFP near the tail of non-Tg, hsp-90(p673) , TDP43 (M337V) , and TDP43 (M337V) ; hsp-90(p673) animals. Scale bars = 10μm. B. TDP-43 (M337V) expressing C . elegans increase rpt-3p :: GFP reporter expression, but hsp-90 mutation does not affect reporter expression either independently or in combination with TDP-43 (M337V) . Error bars represent SEM: N = 3, n = 25–31. Statistical significance as determined using the non-parametric Kruskal-Wallis test (Dunn’s multiple comparisons post hoc test).

Article Snippet: C . elegans β-tubulin levels were measured using monoclonal antibody E7 (Developmental Studies Hybridoma Bank, 1:5000) as a loading control.

Techniques: Expressing, Mutagenesis