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

Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Size of EBs and time (days) in the suspension culture affects the expression of germ layer specific genes. During suspension cell culture, the pluripotency markers, (A) NANOG and (B) SOX2, decreased among all studied variants. Gene expression analysis indicated that the mesoderm, (C) Brachyury and (D) MIXL1, germ layer was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of suspension culture (day 15). The mesenchymal marker (E) SMA and endoderm marker (F) FOXA2 did not predominantly express in the studied variants. Gene expression analysis indicated that the ectoderm germ layer, (G) VIM and (H) PAX6, was favoured in EBs formed from 500 and 1,000 cell wells when compared with the 1,500 and 2,000 cell wells at the end of the suspension culture (day 15). The y-axis represents the relative expression of analysed genes, normalized to the BG01V cell line. Data are presented as the mean ± standard deviation. *P<0.05, as indicated. EBs, embryoid bodies; SOX2, sex determining region Y-box 2; MIXL1, mix paired-like homeobox; α-SMA, α-smooth muscle actin; FOXA2, forkhead box protein A2; VIM, vimentin; PAX6, paired box 6.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Expressing, Cell Culture, Gene Expression, Marker, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Effect of cellular mass on chondrogenic differentiation during embryoid body formation

doi: 10.3892/mmr.2018.9272

Figure Lengend Snippet: Smaller EBs exhibit more prochondrogenic outcomes. Following collection of EBs from suspension culture, the 3 week chondrogenic differentiation protocol was performed and gene expression was analysed. Expression of (A) NANOG and (B) SOX2 decreased in all studied variants. Expression of (C) T, (D) SOX9 and (E) FGFR3 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (F) CD44 was significantly expressed in differentiated 2,000-cell wells EB collected on the fifth day of suspension culture. (G) The low level of COL1A2 expression was observed in all studied variants following chondrogenic differentiation. (H) Expression of COL2A1 was significantly higher in EBs formed in 500-cell wells when compared with 2,000-cell wells. (I) ACAN expression increased only in EBs collected on day 5. Overall, 15 days of suspension culture resulted in a lower expression of analysed genes in chondrogenically differentiated EBs formed from 500- and 2,000-cell wells. The y-axis represents the relative expression of analysed genes, normalized to the BG01V or HC-402-05a cell line. Data are presented as the mean ± standard deviation. *P<0,05, as indicated. EBs, embryoid bodies; hESC, human embryonic stem cells; SOX, sex determining region Y-box; T, brachyury gene; FGFR3, fibroblast growth factor receptor 3; CD44, cluster of differentiation 44; COL, collagen; ACAN, aggrecan.

Article Snippet: The BG01V hESC line (American Type Culture Collection, Manassas, VA; USA) was cultured in mitomycin-C-treated mouse embryonic fibroblasts (MEFs; passage 3; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) seeded on a culture dish previously coated with MatrigelTM (Corning Incorporated, Corning, NY, USA).

Techniques: Suspension, Gene Expression, Expressing, Standard Deviation