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: Scientific reports

Article Title: Zebrafish VCAP1X2 regulates cardiac contractility and proliferation of cardiomyocytes and epicardial cells.

doi: 10.1038/s41598-018-26110-3

Figure Lengend Snippet: Figure 2. VCAP1X2 mutant displayed cardiac dysfunction and impaired cardiomyocyte proliferation. (A) Pseudodynamic reconstructed 3D images and respective bright-field images are shown of end-diastolic phase beating hearts from Tg(myl7:EGFP; myl7:H2AFZ mCherry) (a,b) or VCAP1X2 mutant (c,d) at 72 hpf. Yellow lines indicate the diameter of the ventricular chamber. Green fluorescence marks ventricular myocardium and red indicates ventricular chamber. Scale bar, 50 μm. (B) Parameters related to heart performance were measured from pseudodynamic reconstructed 3D images (a–c, n = 3, N = 2) or CCD images (d–f, n = 20, N = 3) for Tg(myl7:EGFP; myl7:H2AFZ mCherry) or WT and VCAP1X2 mutant at 72 hpf. End-systolic volume (a) and end-diastolic volume (b) were significantly increased in VCAP1X2 mutant compared with Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos. Ejection fraction (c) was lower in VCAP1X2 mutant than in Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos. End-diastolic length (d) and width (e) were increased while fractional shortening (f) was decreased in VCAP1X2 mutant compared to WT. Error bars represent standard error. Student’s t-test, **p < 0.01, ***p < 0.001. (C) Paraffin sectioning with hematoxylin and eosin staining revealed a thinner compact layer in the ventricle of VCAP1X2 mutants (b) and LacZ mRNA-injected VCAP1X2 mutants (c) compared to WT (a) or VCAP1X2 mRNA-injected VCAP1X2 mutants (d) (n = 10 per condition). a, atrium, v, ventricle. Scale bar, 50 μm. (D) Immunostaining of BrdU (red) in heart ventricles from Tg(myl7:EGFP; myl7:H2AFZ mCherry) (myl7) (a) or VCAP1X2 mutants (b) at 72 hpf. Significantly decreased cardiomyocyte (CM) number (c) and percentage of BrdU+ CMs (d) in the ventricle was detected in VCAP1X2 mutant compared to Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos (n = 20 per condition, N = 3). Student’s t-test, ***p < 0.001. Scale bar, 50 μm. Apoptotic cells were analyzed by TUNEL labeling (E) and similar low level of apoptotic cells (F) was detected in VCAP1X2 mutant and Tg(myl7:EGFP; myl7:H2AFZ mCherry) embryos (n = 15, N = 3). Cardiomyocyte nuclei stained by anti-MEF2 antibody. Error bars represent standard error. Scale bar, 50 μm.

Article Snippet: Tissues were incubated with primary antibody including MEF2 (1:150, Santa Cruz Biotechnology) or PCNA (1:200, Abcam) at 4 °C overnight.

Techniques: Mutagenesis, Fluorescence, Staining, Injection, Immunostaining, TUNEL Assay, Labeling

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: The p38 pathway and Mef2D are rate-limiting for late-stage genes. (A) Examples of simple network motifs based on Lee et al. (2002). In a single-input motif, factor A directly regulates the three targets B, C, and D. The simple cascade depicts sequential activation with only gene B directly activated by A. In the feed-forward loop, A directly regulates each gene and sequential activation is achieved by requiring both A and B to express gene C, and both A and C to express gene D. (B) MDER fibroblasts were induced to differentiate for varying times and analyzed by Northern and Western blot. (SB) Treatment of cells with SB203580; (CIP) phosphatase treatment prior to SDS-PAGE. (C) Ratio of the average gene expression in each temporal cluster of MyoD-activated genes comparing cells constitutively expressing MKK6E or treated with SB203580 with the expression in untreated cells, demonstrating effects of p38 activity on clusters of genes activated at different stages of differentiation. Ratios are in log2 space; a value of 0 indicates no treatment change, a value of 1 indicates a twofold increase due to the treatment, and a value of -1 indicates a twofold reduction. Microarray expression data was generated from MDER cells induced to differentiate for 24 h with MKK6E-expressing virus or control virus, and analyzed together with previously generated data for control and SB203580-treated MDER cells (Bergstrom et al. 2002). Clusters 1-6 are the earliest to latest activated clusters, and cluster 7 is a transiently expressed group of genes (Bergstrom et al. 2002). Error bars represent standard error, except cluster 7, where error bars are absent because only one gene from this cluster was affected. ANOVA p-value < 0.0001 for both MKK6E and SB203580. Post-hoc testing indicated that cluster 6 differs significantly from the other clusters in response to MKK6E. (D) MDER cells were infected with the indicated retroviruses, induced to differentiate for 12 h, and subjected to Northern analysis for late-stage genes Myh3 and Des and the early-stage gene Cdh15. Mef2 is the Mef2D isoform, Con is an empty control retrovirus.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Activation Assay, Northern Blot, Western Blot, SDS Page, Expressing, Activity Assay, Microarray, Generated, Infection

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: Mef2D and p38 are rate limiting for the activation of late-stage genes. (A) MDER cells were infected with MKK6E and/or Mef2D-expressing retrovirus as indicated, induced to differentiate for varying lengths of time, and analyzed by Northern blot with the indicated probes. GFP is used as a control retrovirus. (B) MDER fibroblasts were transfected with the MKK6E and/or Mef2D retrovirus as indicated, induced to differentiate, and examined by immunofluorescence for the indicated late-stage (Myh3 and Des) and early-stage (Mgn) gene products. The bars indicate the average number of positive cells per field.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Activation Assay, Infection, Expressing, Northern Blot, Transfection, Immunofluorescence

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: p38 regulates transcription factor recruitment. (A) MDER fibroblasts were either untreated (0 h) or infected with MKK6E retrovirus (+) or empty vector control retrovirus (-) and induced to differentiate for 12, 24, or 48 h. ChIP was performed with broad-specificity Mef2 antiserum, and multiplex PCR for the indicated promoters was performed with pancreatic amylase (Amy) as an internal control. Input chromatin was amplified over a 30-fold range to verify linearity of the assay. Graphs indicate IP promoter fold enrichment relative to input chromatin. Data for a representative ChIP are shown with error bars indicating S.E.M. for triplicate PCR. (B) Mef2 ChIP demonstrating that active MyoD is necessary for Mef2 binding, and that the broad-specificity Mef2 antisera identifies Mef2 binding both with and without exogenous Mef2D expression. (NS) Nonspecific IgG control ChIP; (E2) β-estradiol to induce MyoD activity. (C) ChIP using Mef2D monoclonal antibody shows Mef2D is not bound at early times in the absence of exogenous Mef2D. (GFP) A control retrovirus; (K6 + 2D or 6 + D) a combination of the MKK6E and Mef2D retroviruses. (D) ChIP using MyoD antisera shows p38 regulation of MyoD binding but no dependence on Mef2D. Input titration shown in C. (Empty) Control virus without effector gene.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Infection, Plasmid Preparation, Multiplex Assay, Amplification, Binding Assay, Expressing, Activity Assay, Titration

Journal:

Article Title: A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation

doi: 10.1101/gad.1234304

Figure Lengend Snippet: Mef2D and p38 regulate Pol II recruitment and progression. (A) MDER cells were transduced with control (GFP), MKK6E (K6), and/or Mef2D (2D) retroviruses, induced to differentiate 12 h and ChIP for Pol II, or P-CTD Pol II performed. The combination of Mef2D and p38 is associated with Pol II recruitment and phosphorylation. (B) MDER cells were transduced with the indicated retroviruses and induced to differentiate for 16 h. At this later time point when endogenous Mef2D and other muscle-specific Mef2 isoforms are accumulating, p38 activation induces polymerase progression and accumularion of P-CTD Pol II in the 3′ end of the gene. ChIP performed for Pol II and Ser 5-phosphorylated Pol II (P-CTD Pol II). Amplification of the gene promoter and a region in the 3′-transcribed regions of the gene (exon 7 of desmin, 6.5 kb from the promoter, and exon 27 of MHC, 19 kb from the promoter) was performed. Titrations, internal control, and graphs are as previously indicated.

Article Snippet: ChIP, Western, immunofluorescence, and antibodies Antibodies used for ChIP were as follows: Mef2A (Santa Cruz), note that this antibody also recognizes Mef2C and Mef2D (B.H.

Techniques: Transduction, Activation Assay, Amplification