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: International Journal of Biological Sciences

Article Title: Cdh1 functions as an oncogene by inducing self-renewal of lung cancer stem-like cells via oncogenic pathways

doi: 10.7150/ijbs.38672

Figure Lengend Snippet: Primers for RT-qPCR

Article Snippet: The following primary antibodies were used: anti-E-cadherin (Proteintech, USA), anti-PI3K, anti-Akt, anti-mTOR, anti-S6K, anti-Eif4e, anti-Erk1/2, anti-p-Mek1/2, anti-p-Erk1/2, anti-p-Akt, anti-p-mTOR, anti-p-S6k (Cell Signaling Technology, USA), anti-Mek1 (Upstate, USA) and anti-GAPDH (Proteintech, USA).

Techniques:

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: Journal of Biological Chemistry

Article Title: A potential target for liver cancer management, lysophosphatidic acid receptor 6 (LPAR6), is transcriptionally up-regulated by the NCOA3 coactivator

doi: 10.1074/jbc.ra119.009899

Figure Lengend Snippet: Fig. 5 NCOA3 regulates H3K27ac enrichment at LPAR6 locus (A) Schematic diagram of LPAR6 gene and specific primers designed for qPCR. Black box represents CDS region of LPAR6; Gray boxes represent amplification sites by ChIP-qPCR. (B-C) H3K27ac enrichment in (B) HepG2 or (C) Huh7 cells with or without NCOA3 knockdown. Equal amounts of cells were collected after NCOA3 shRNA or scrambled shRNA transfection for 48h and then subject to ChIP. Primers designed for Site 1 amplified LPAR6 coding sequence, while Site 2 and 3 amplified LPAR6 promoter. Error bars represent S.D. from three independent biological replicates. *p< 0.05 and **p< 0.01, significance of difference was analyzed by unpaired t-test. (D) HGF treatment decreased NCOA3 expression in HepG2

Article Snippet: Antibody against NCOA3 (20032-1-AP) was purchased from Proteintech.

Techniques: Amplification, ChIP-qPCR, Knockdown, shRNA, Transfection, Sequencing, Expressing