Journal: Science Advances

Article Title: BRD4770 protects against DOX-induced cardiotoxicity by inhibiting apoptosis and ferroptosis

doi: 10.1126/sciadv.adw1720

Figure Lengend Snippet: ( A ) Schematic of cell-based high-throughput screening of epigenetic compounds for protection against DOX/erastin-induced cardiomyocyte death. ( B ) The scatter plot of the effect of epigenetic compounds on DOX (top)– or erastin (bottom)–induced cardiomyocyte death. ( C ) Venn analysis of a drug compound. The top 25 compounds that protect against DOX- or erastin-induced cardiomyocyte death were selected separately, overlapping a total of 8 compounds. ( D ) The eight compounds that protect against both DOX- and erastin-induced cardiomyocyte death. Red indicates compounds that have been studied, and green indicates compounds that have not yet been reported in DIC. BDNF, brain-derived neurotrophic factor; JNK1, c-Jun N-terminal kinase 1; TrkA, tropomyosin receptor kinase A; Pim1, proviral integration site for Moloney murine leukemia virus-1 (Pim1) kinase; EGFR, epidermal growth factor receptor; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; HDAC1, histone deacetylase 1. ( E ) The effects of J147 (5 μM), BRD4770 (5 μM), SCR7 pyrazine (5 μM), SMI-4a (5 μM), daphnetin (5 μM), BG45 (5 μM), and entinostat (5 μM) on cell viability in H9C2 cells after 24 hours of DOX or erastin treatment ( n = 6). Statistical significance was determined using (E) one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. * P < 0.05 and ** P < 0.01.

Article Snippet: BRD4770 (#T1923), J147 (#T1993), BG45 (#T2294), SMI-4a (#T3058), daphnetin (#T2851), entinostat (#T6233), ISRIB (#T2027), OTS186935 (# T12344 ), and UNC0638 (#T3257) were purchased from TargetMol (Wellesley Hills, MA, USA).

Techniques: High Throughput Screening Assay, Derivative Assay, Virus, Histone Deacetylase Assay

Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: Nuclear kinases that phosphorylate N-terminal regions of LANA.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques:

Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: A. Phosphorylation of a LANA aa3–21 peptide by the indicated kinases. The peptide was separated from the kinase reaction mixture by electrophoresis and the gel dried and subjected to autoradiography. B. Upper: Amino acid sequence of the wt LANA 3–21 peptide (1) showing the positions of the single and grouped sequence changes in the mutant peptides (2–6). Lower: Examples of phosphorylation of the wild-type and mutant peptides by the indicated kinases. C. GSK-3 phosphorylates S10 after PIM1 priming. Wild-type and S10A mutant peptides were incubated with PIM1 or GSK-3β or with PIM1 plus unlabelled γ-ATP (PIM1 priming) or with GSK-3 β after PIM1 priming followed by inhibition of PIM1 with the PIM1 inhibitor SMI-4a. (−), minus kinase.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques: Electrophoresis, Autoradiography, Sequencing, Mutagenesis, Incubation, Inhibition

Journal: PLoS Pathogens

Article Title: Phosphorylation of the Chromatin Binding Domain of KSHV LANA

doi: 10.1371/journal.ppat.1002972

Figure Lengend Snippet: A. Cells treated with CKI inhibitor (CKI-7). B. Cells treated with GSK-3 inhibitor (LiCl) or PIM1 inhibitor (SMI-4a) individually or in combination. C. Cells treated with RSK inhibitor (BRD 7389). Western blots examine interaction between transfected Flag-LANA and endogenous histone H2B. Quantitation of the band densities provides a measure of relative binding (LANA bound H2B/input H2B) and was obtained using Image J. The ratio in untreated cells was set at 100%.

Article Snippet: For characterization of GSK-3β phosphorylation, peptide substrates were incubated for 30 min at 30°C in (i) kinase buffer with 10 µCi of [γ 32 P]-ATP, (ii) kinase buffer with 10 µCi of [γ 32 P]-ATP and Pim1 (Upstate, #14-573), (iii) kinase buffer with 10 µCi of [γ 32 P]-ATP and GSK-3β (Millipore) and for primed phosphorylations (iv) kinase buffer with 20 µM cold ATP and Pim1 (Upstate, #14-573) followed by incubation with Pim1 inhibitor SMI-4a (0.1 µM; Enzo Life Sciences) for 30 min at 30°C (Millipore).

Techniques: Western Blot, Transfection, Quantitation Assay, Binding Assay