Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: Crystal structure of SMYD2 in complex with LLY-507. A , SMYD2 is depicted as thin sticks in green , whereas the bound LLY-507 and S -adenosyl- l -homocysteine ( SAH ) are depicted as thick sticks in magenta and cyan , respectively. The F o − F c omit map electron density of LLY-507 in blue is contoured at 3σ. B , plot of SMYD2 interactions with LLY-507. C , structure of SMYD2 in complex with mono-methylated p53 peptide (Ref. ; Protein Data Bank code 3S7D ) with SMYD2 depicted as thin sticks in orange , and S -adenosyl- l -homocysteine and p53 peptide depicted as thick sticks in cyan and magenta , respectively. D , crystallographic and refinement statistics for the structure of SMYD2 in complex with LLY-507. R.m.s. , root mean square.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: Chemical structure and biochemical profile of LLY-507. A , chemical structure of LLY-507. B , effect of LLY-507 on the methyltransferase activity of SMYD2 using p53(361–380) peptide as a substrate (IC 50 < 0.015 μ m ). Error bars represent S.D. C , thermostability melting curves of SMYD2 in the absence ( red ) or presence ( green ) of 100 μ m LLY-507. D , effect of LLY-507 at 1 ( light gray ), 10 ( dark gray ), and 50 μ m ( black ) against SMYD2 and 25 other protein or DNA methyltransferases.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: LLY-507 inhibits SMYD2-mediated methylation of p53 Lys 370 in cells. A , Western blot showing concentration-dependent inhibition of p53 Lys 370 me1 following treatment with 0–2.5 μ m LLY-507 in HEK293 cells transiently transfected with FLAG-SMYD2 and FLAG-p53 (IC 50 < 1 μ m ). B , cell-based ELISA for p53 Lys 370 me1 of U2OS cells transfected with SMYD2 following treatment with LLY-507 for 15 h (IC 50 = 0.6 μ m ). C , concentration-dependent inhibition of p53 Lys 370 me1 by LLY-507 in KYSE-150 cells stably expressing SMYD2 as measured by a Meso Scale Discovery sandwich ELISA (IC 50 = 0.6 μ m ). D , Western blot showing a time-dependent decrease in p53 Lys 370 mono-methylation with 5 μ m LLY-507 treatment of KYSE-150 cells stably expressing FLAG-SMYD2. E , Western blot examining the protein levels of p53-Lys 370 me1 and p53 as well as α-tubulin, RNA polymerase ( pol ) II, and histone H2AX positive controls for cytoplasmic, soluble nuclear, and chromatin subcellular fractions, respectively, in KYSE-150 cells stably expressing FLAG-SMYD2. F , effect of LLY-507 on SMYD2 biochemical activity using histone H4(1–24) peptide as substrate (IC 50 = 31 n m ). G , relative intensities of methylated histone H3 or H4 peptides in SMYD2-overexpressing KYSE-150 cells treated with vehicle or 5 μ m LLY-507, identified and quantified using mass spectrometry. A full list of histone H3 and H4 peptides and their intensities is found in supplemental Table 5 . H , Western blot examining the protein levels of endogenous SMYD2 as well as α-tubulin, RNA polymerase ( Pol ) II, and histone H2AX positive controls for KYSE-150 cytoplasmic, soluble nuclear, and chromatin subcellular fractions, respectively. un , un-methylated; me1 , mono-methylated; me2 , di-methylated; me3 , tri-methylated; ac , acetylated.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Methylation, Western Blot, Concentration Assay, Inhibition, Transfection, In-Cell ELISA, Stable Transfection, Expressing, Sandwich ELISA, Activity Assay, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: LLY-507, a Cell-active, Potent, and Selective Inhibitor of Protein-lysine Methyltransferase SMYD2 *

doi: 10.1074/jbc.M114.626861

Figure Lengend Snippet: LLY-507 inhibits the proliferation of several ESCC, HCC, and breast cancer cell lines. A , effect of 3- or 7-day treatment with LLY-507 on the proliferation of ESCC, HCC, and breast cancer cell lines. Cells were treated for either 3–4 or 7 days with compound, and cell viability was then measured using CellTiter-Glo. B , concentration-response curves depicting effect of LLY-507 on 3- and 7-day growth of KYSE-150 cells. C , summary of the status of p53 or Rb in the ESCC, HCC, and breast cancer cell lines tested for the antiproliferative effects of LLY-507 derived from mining of the Cancer Cell Line Encyclopedia. D , mRNA expression analysis of hADR-α1A and SMYD2 in cell lines used in this study derived from mining of the Cancer Cell Line Encyclopedia. LOH , loss of heterozygosity.

Article Snippet: SULFO-TAG TM total p53 detection antibody was added to the plate at a final concentration of 1.5 mg/ml in Antibody Dilution Buffer (Meso Scale Discovery) followed by shaking for 1 h at room temperature.

Techniques: Concentration Assay, Derivative Assay, Expressing

Journal: PLoS ONE

Article Title: Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

doi: 10.1371/journal.pone.0104639

Figure Lengend Snippet: Significant dose-responses of PCB 180 based on total dose.

Article Snippet: The primary antibody for total p53 DO-1 was purchased from Novocastra (Newcastle, UK).

Techniques: Cell Counting, Concentration Assay, Activity Assay

Journal: PLoS ONE

Article Title: Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

doi: 10.1371/journal.pone.0104639

Figure Lengend Snippet: Significant dose-responses of PCB 180 based on adipose tissue concentration.

Article Snippet: The primary antibody for total p53 DO-1 was purchased from Novocastra (Newcastle, UK).

Techniques: Concentration Assay, Cell Counting, Activity Assay

Journal: PLoS ONE

Article Title: Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

doi: 10.1371/journal.pone.0104639

Figure Lengend Snippet: Tumor suppressor protein p53 and the DNA damage signaling markers were dose-dependently increased only in females. Each column represents mean ± SD (n = 5) as percent of control after adjustment to the loading control (Cdk2). Data was obtained from at least three independent analyses.

Article Snippet: The primary antibody for total p53 DO-1 was purchased from Novocastra (Newcastle, UK).

Techniques: Control

Journal: PLoS ONE

Article Title: Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

doi: 10.1371/journal.pone.0104639

Figure Lengend Snippet: Margins of exposure (MoE = CED-L/human median PCB 180 concentration) for different endpoints of PCB 180 toxicity and different human cohorts. MoE values exceeding the WHO default uncertainty factor of 25 are bolded.

Article Snippet: The primary antibody for total p53 DO-1 was purchased from Novocastra (Newcastle, UK).

Techniques: Concentration Assay, Activity Assay