Journal: bioRxiv

Article Title: Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy

doi: 10.1101/718833

Figure Lengend Snippet: A. Median inhibitory concentration (IC 50 ) values for NDGA on different acetylated histone residues and median effective concentration (EC 50 ) value on H3K27 tri-methylation were determined by immunoblotting of histones extracted from cells after 24-hour treatments with varying concentrations of NDGA (ND, not determined). B. Differences in acetylation and tri-methylation of p300 target (H3K14, H3K18, H3K27) and non-target (H3K9) histones were monitored by immunoblotting after indicated concentrations of NDGA treatment for 24 hours and histone extraction. C. Dose-dependent changes in acetylation and tri-methylation on H3K27 after 24-hour NDGA treatment were analyzed by immunoblotting and are represented as percentage change in histone modification relative to DMSO treatment. D. Decreases in H3K27 acetylation upon 30 μM of NDGA treatment for 24 hours in human (HEK293T), mouse (MEF) and fruit fly (S2 Schneider) cells were monitored by immunoblotting after histone extraction. E. Effects of wildtype p300 overexpression on NDGA-dependent H3K27 hypoacetylation were determined by densitometric analysis of immunoblotting data. Cells were transfected with different amounts of human wildtype EP300 encoding expression plasmid pCI-p300(WT) and/or empty vector (EV) and treated with varying NDGA concentrations for 24 hours. The data represent percentage change in H3K27 acetylation relative to DMSO treatment and the change in IC 50 values. F. The rescue of p300 overexpression on H3K27 hypoacetylation after 30 μM NDGA treatment was performed as in panel E. G. Effects of overexpression of wildtype and catalytically inactive p300 mutants (Y1503A and F1504A) on NDGA-induced H3K27 hypoacetylation were determined by immunoblotting and densitometry. Cells were transfected by the same amount of expression plasmids or empty vector (EV) and treated with varying NDGA concentrations. Histones were extracted and analyzed. H. The effects of wildtype and mutant (Y1503A or F1504A) p300 overexpression on NDGA-induced histone hypoacetylation and hypermethylation were determined by immunoblotting after 30 μM NDGA treatment for 24 hours. I. Effects of wildtype and mutant p300 overexpression on NDGA-induced histone hypoacetylation were analyzed by immunoblotting and densitometry. J. Decreased H3K27 tri-methylation upon wildtype p300 overexpression was detected by immunoblotting and densitometry. Total histone antibodies were used as loading controls in all experiments. Data represent the results from two independent experiments (mean ± S.D.) and curves were generated using nonlinear regression fit.

Article Snippet: HEK293T (ATCC), HeLa-LC3-GFP and MEF (ATCC) cells were maintained in appropriate volume of Dulbecco’s Modified Eagle Medium supplemented with 10% serum (Serum Plus - II, Sigma), 100 units/mL penicillin and 100 units/mL streptomycin (Corning, VA).

Techniques: Concentration Assay, Methylation, Western Blot, Extraction, Modification, Over Expression, Transfection, Expressing, Plasmid Preparation, Mutagenesis, Generated