Journal: Scientific Reports

Article Title: Histone acetylation determines transcription of atypical protein kinases in rat neurons

doi: 10.1038/s41598-019-40823-z

Figure Lengend Snippet: Blockade of translation with anisomycin (Ani, 10 μM) selectively altered the TSA-induced regulation of PKMζ and PKCζ expression, while epigenetically-driven upregulation of PKCλ expression was unaffected. ( a ) Application of Ani did not impair the TSA-induced upregulation of PKCλ expression. Control, TSA-treated (19 h) cultures and cultures treated with Ani alone (20 h) or in combinations with TSA were used for comparisons. For the last group, Ani was administered for 1 h before the application of TSA. [F (3,17) = 20,913; p < 0,001; n = 5–6/group]. ( b , c ) Ani prevented downregulation of PKMζ expression in TSA-treated cultures [KW: H (3, N = 24) = 13,22000 p = 0,0042; *p = 0,002; & p < 0,001; n = 6/group] and significantly alleviated epigenetically-driven upregulation of PKCζ expression [KW: H (3, N = 20) = 16,45893 p = 0,0009; *p < 0,001; n = 5/group]. Representative experiment below demonstrates patterns of PKCζ expression in the current experimental series. mRNA levels were determined by standard PCR followed by agarose gel electrophoresis. YWHAZ served as a reference. *Significant differences; & Significant difference (relative to standard control group); ns – not significant.

Article Snippet: Transcription inhibitor actinomycin D (ActD, final concentration 200 nM, 4 μM; Sigma-Aldrich), and protein synthesis inhibitor anisomycin (Ani, final concentration 10 μM, 100 μM; Tocris) were used for verification of experiments with histone deacetylase inhibitors.

Techniques: Expressing, Control, Agarose Gel Electrophoresis

Journal: The American journal of pathology

Article Title: HIV Protease Inhibitors Alter Amyloid Precursor Protein Processing via β-Site Amyloid Precursor Protein Cleaving Enzyme-1 Translational Up-Regulation.

doi: 10.1016/j.ajpath.2016.09.006

Figure Lengend Snippet: Figure 6 Ritonavir up-regulates neuronal BACE1 expression via translation-dependent control. A: Rat neurons were exposed to indicated treatments for 16 hours. BACE1 mRNA was determined using the DDCt method relative to TATA-Box binding protein (TBP). Quantitative RT-PCR assays were performed in triplicate (one-way analysis of variance, Dunnett post hoc). B: Rat neurons were either treated with ritonavir alone or pretreated with anisomycin or acti- nomycin D for 12 hours before ritonavir. Fast green was used as a loading control. C: Compared to ritonavir-only treated neurons, anisomycin pretreatment resulted in decreased BACE1 expression (one-way analysis of variance, Newman-Keuls post hoc). D: Rat neurons were treated for 16 hours, and cytoplasmic and nuclear fractions were analyzed by immunoblotting. E: Densitometric analysis revealed a significant increase in activating transcription factor 4 (ATF4) and BACE1 (one-way analysis of variance, Dunnett post hoc). F: Fourteen DIV rat cortical cultures were transfected with pcDNA3.1Zeoþ vector containing full human BACE1 coding region, including the BACE1 50 UTR but missing the 30 UTR (þ50 UTR), or pcDNA3.1Zeoþ vector containing human BACE1 coding region, including the 30 UTR but missing the 50 UTR (-50 UTR). Forty-eight hours later, cultures were treated with dimethyl sulfoxide or 10 mmol/L ritonavir for 16 hours, and immunoblotted for BACE1. Representative blots are shown. G: Quantification of BACE1 was normalized to actin (one-way analysis of variance, Newman-Keuls post hoc). H: Rat neurons were treated with 10 mmol/L ritonavir, 1 mmol/L saquinavir, or 10 mmol/L lactacystin for 16 hours. Whole cell lysate (10 mg) from each condition was used to determine 20S proteasome activity (one-way analysis of variance, Dunnett post hoc). n Z 4 (A, C, and E); n Z 3 (B); n Z 2 (H). *P < 0.05, **P < 0.01. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; UT, untreated.

Article Snippet: The following chemical reagents used in the study were purchased from the indicated vendors: Citifluor, Ltd (London, UK): citifluor AF1; BioRad (Hercules, CA): bradford protein assay dye, polyvinylidene difluoride membrane, and prestained broad-range molecular weight ladder; Jackson ImmunoResearch Labs (West Grove, PA): all dye-conjugated secondary antibodies; Life Technologies (Carlsbad, CA): Dulbecco’s modified Eagle’s medium, neurobasal media, and B27 supplement; Millipore (Temecula, CA): 20S proteasome activity assay; Nanotherics (Newcastle under Lyme, UK): neuromag transfection reagent; Sigma (St. Louis, MO): DAPI, fast green FCF, protease inhibitor cocktail, 4-hydroxytamoxifen (H7904), anisomycin (A9789), and actinomycin D (A1410); Peptides International (Louisville, KY): poly-L-lysine; New England Biolabs (Ipswich, MA): tyramide amplification system; Scytek Labs (Logan, UT): normal antibody diluent; Thermo Fisher Scientific (Waltham, MA): SuperSignal West Dura extended-duration substrate and all horseradish peroxidaseeconjugated secondary antibodies.

Techniques: Expressing, Control, Binding Assay, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Activity Assay