Journal: Biomedicines

Article Title: R -α-Lipoic Acid and 4-Phenylbutyric Acid Have Distinct Hypolipidemic Mechanisms in Hepatic Cells

doi: 10.3390/biomedicines8080289

Figure Lengend Snippet: PBA enhances the acetylation of CBP/p300 and histone H3 at the CPT1A and INSIG2 promoters in Huh7-shTSC2 cells. PBA (8 mM, 6 h) induced the gene expression of CPT1A and INSIG2 ( A ), and the acetylation status of histone H3 and histone H4 ( B ). Chromatin immunoprecipitation (ChIP) assay revealed PBA-mediated enrichment of acetyl CBP/p300 (Ac-p300) and acetyl histone H3 (Ac-H3) at the CPT1A and INSIG2 promoters ( C ). Statistical significance was determined using Student’s t -test, * p < 0.05, n = 4.

Article Snippet: After pre-clearing the sheared chromatin, samples were subjected to immunoprecipitation using the following the antibodies: anti-acetyl-CBP [Lys1535]/p300 [Lys1499] (Cell Signaling Technology #4771S, Danvers, MA, USA), anti-acetyl-histone H3 (Millipore #06–599, Burlington, MA, USA), anti-histone H3 (Millipore #07–690).

Techniques: Gene Expression, Chromatin Immunoprecipitation

Journal: Nature Communications

Article Title: Assay interference and off-target liabilities of reported histone acetyltransferase inhibitors

doi: 10.1038/s41467-017-01657-3

Figure Lengend Snippet: Reported HAT inhibitors cause nonspecific cell assay readouts. a Double p300 and CBP knockdowns do not affect cell viability after 2 d. Data are expressed as mean ± SEM of three technical replicates (ns, not significant; two-tailed Student’s t -test). b Double p300 and CBP knockdowns efficiently reduce H3K27ac levels in HEK293T and MCF7 cells. Molecular weight of probed protein is indicated in kilodaltons (kDa) as verified by molecular weight markers. Data are representative results from one of two independent experiments. c Many reported HAT inhibitors exert anti-proliferative effects at low micromolar compound concentrations in (top) MCF7 and (bottom) HEK293T cells as monitored by cell confluence 24 h after addition of compound. Note the nonspecific interference compounds NSC-663284 (N), rottlerin (R), and 24 – 27 (chemical structures shown as inset) demonstrate similar anti-proliferative effects. C, DMSO control. Red error bars, statistically significant difference from respective DMSO controls ( p < 0.05; two-tailed Student’s t -test and Holm–Sidak method). Data are expressed as mean ± SD of three technical replicates and are representative results from one of two independent experiments. d Many reported HAT inhibitors also cause nonspecific changes in H3K27ac and p300 levels in cells at low micromolar compound concentrations. Molecular weights of protein analytes are indicated in kDa as verified by molecular weight markers (Supplementary Fig. ). Note the same known interference compounds from panel c can also decrease H3K27ac levels. Data are representative results from one of two independent experiments

Article Snippet: Blots were blocked for 1 h in blocking buffer (5% milk in 0.1% Tween 20/PBS) and incubated with respective primary antibodies: H3K27ac (1:1000, CST #8173), H3 (1:1000, Abcam #10799), p300 (1:2000, Bethyl #A300-358A), CBP (1:1000, Bethyl #A300-362A), actin (1:3000, Abcam #3280), α-tubulin (1:1000, 12G10 DHSB), and α-tubulin-K40ac (1:2000, Abcam #179484) in blocking buffer overnight at 4 °C.

Techniques: Two Tailed Test, Molecular Weight, Control

Journal: Journal of cell science

Article Title: Promoter occupancy of MLL1 histone methyltransferase seems to specify the proliferative and apoptotic functions of E2F1 in a tumour microenvironment.

doi: 10.1242/jcs.126235

Figure Lengend Snippet: Fig. 8. Mutually exclusive promoter occupancy of E2F1 co-activator/repressor complexes in hepatoma cells. (A) Liver tissue lysates from 3- and 6-month-old C57B6 control (C) and X15-myc transgenic (T) mice were immunoprecipitated with anti-E2F1 and immunoblotted for CBP, GCN5 and Brg1. One-tenth of the tissue lysate was used to probe the expression of GCN5 and HBx. Actin was used as loading control. (B–E) HepG2.2.15 cells were transfected with either scrambled (scr) siRNA or CBP-specific siRNA. Cells were serum starved 24 hours post-silencing and subjected to serum stimulation (SS) either in the absence or presence of cisplatin (SS + cisplatin) and processed for ChIP-qPCR analysis. Enrichment of CBP and Brg1 occupancy over IgG control was measured on either MCM4 (B) or Apaf-1 gene promoters (D). Similarly, the same samples were used to measure the enrichment of MLL1 and E2F1 occupancy over IgG control on either MCM4 (C) or Apaf-1 gene promoters (E). Data are means 6 s.d. of three independent experiments; *P,0.05 and #P,0.01.

Article Snippet: For RNA interference studies, 2.0 mg of HBx shRNA (X-D) was transfected using Fugene 6 (Invitrogen). siRNA against E2F1, MLL1 and CBP (all from Santa Cruz Biotechnology) were used for transfection according to manufacturer’s instructions.

Techniques: Control, Transgenic Assay, Immunoprecipitation, Expressing, Transfection, ChIP-qPCR

Journal: PLoS Pathogens

Article Title: Myeloid neddylation targets IRF7 and promotes host innate immunity against RNA viruses

doi: 10.1371/journal.ppat.1009901

Figure Lengend Snippet: (A)Twenty-four hours after HEK-293T cells were transfected with mammalian expression vectors encoding GFP-tagged murine IRF7 WT and K3R mutant, the cells were infected with SeV for 6 h or left untreated. Then the subcellular localization of exogenous murine IRF7 was examined via confocal microscopy. (B) After BMDMs were cultured from Nedd8 F/F and Nedd8 ΔMye mice, the cells were infected with SeV for 6 h or left untreated. The subcellular localization of IRF7 was examined by nuclear cytoplasmic fractionation and subsequent IB. GAPDH was regarded as a cytoplasm marker and H3 as a nucleus marker. (C, D) Twenty-four hours after HEK-293T cells were transfected with the indicated mammalian expression vectors, the cells were infected with SeV for 6 h or left untreated. Then the interaction between exogenous murine IRF7 and exogenous murine CBP was determined by Co-IP (C). The specific binding of exogenous murine IRF7 to selected Ifna gene promoters within the chromatin was analyzed by ChIP with an anti-Myc antibody (D, Top ). Immunoprecipitated exogenous murine IRF7 was analyzed by IB analysis (D, Bottom ). Quantitative data are shown as Mean ± SD ( n = 3 per group). * p < 0.05; *** p < 0.001.

Article Snippet: Mammalian expression vectors encoding FLAG-Myc-tagged murine IRF7 (Cat. No. MR225814), FLAG-tagged human IRF3 (Cat. No. HG12007), FLAG-tagged murine CBP (Cat. No. 32908), and FLAG-tagged human IRF5 (Cat. No. CH890176) were obtained from Origene (Rockville, MD, USA), Sino Biological Inc. (Beijing, China), Addgene (Watertown, MA, USA) [ ], and Vigenebio (Jinan, Shandong, China), respectively.

Techniques: Transfection, Expressing, Mutagenesis, Infection, Confocal Microscopy, Cell Culture, Fractionation, Marker, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation