Journal: bioRxiv

Article Title: A molecular mechanism by which amyloid-β induces inactivation of CREB in Alzheimer’s Disease

doi: 10.1101/2020.01.08.898304

Figure Lengend Snippet: (A) Scheme representing the timeline of the experiment. Hippocampal cultures were silenced with TTX overnight and subsequently treated with forskolin, CNQX, and nifedipine. The time of NMDA bath application was 2, 5, 10 or 15 minutes. (B) Representative immunoblot of total lysates probed with antibodies detecting pCREB and anti-Histone 3 as a loading control. Hippocampal neurons (DIV6) were infected with lentivirus carrying shRNA-NLS-GFP construct or scrambled control. At DIV15 neurons were treated as depicted in the scheme A. (C) Nuclear Jacob knockdown partially rescues CREB shut-off after 5, 10 and 15 minutes of treatment with NMDA. Bar plot represents quantification of pCREB band intensity normalized to Histone 3. Data are represented as mean ± SEM. n=4 from independent experiments. n.s.- non-significant where p>0,05, *p<0,05, **p<0,01 by two-tailed Student t-test.

Article Snippet: Briefly, DIV7 cultures were infected with lentivirus carrying Jacob knockdown or scrambled construct for 12 h. At DIV16, cells were silenced overnight with 1 μM tetrodotoxin (TTX, Carl Roth) and on the following day after TTX washout cultures were treated with 100μM Forskolin (Biorbyt), 10μM Nifedipine (calcium channel blocker, Santa Cruz), and 40μM CNQX (Tocris).

Techniques: Western Blot, Control, Infection, shRNA, Construct, Knockdown, Two Tailed Test

Journal:

Article Title: Cathepsin-L, a Key Molecule in the Pathogenesis of Drug-Induced and I-Cell Disease-Mediated Gingival Overgrowth

doi:

Figure Lengend Snippet: Latent and active cathepsin activity of cells cultured with or without nifedipine. Enzyme activities of active forms of cathepsin-(B+L) (A), active form of cathepsin-B (B), total forms of cathepsin-(B+L) (C), and total form of cathepsin-B (D) were measured in gingival fibroblasts cultured with or without 100 ng/ml of nifedipine. Gingival fibroblasts cultured with or without nifedipine were subjected to measurement of cathepsin activities as described in Materials and Methods. Values are representative of the results obtained from gingival fibroblasts isolated from normal gingival tissues and nifedipine-induced gingival overgrowth, since gingival fibroblasts obtained from nifedipine-induced gingival overgrowth and from normal tissues were similarly affected by nifedipine. Values are expressed as the % activity against that of controls (day 0). Both active and total forms of cathepsin-(B+L) activity were significantly suppressed by nifedipine, while that of cathepsin-B was not influenced.

Article Snippet: Reagents The blocker of L-type Ca (2+) channels, nifedipine, was from Bayer AG Pharma Research Center (Wuppertal, Germany).

Techniques: Activity Assay, Cell Culture, Isolation

Journal:

Article Title: Cathepsin-L, a Key Molecule in the Pathogenesis of Drug-Induced and I-Cell Disease-Mediated Gingival Overgrowth

doi:

Figure Lengend Snippet: mRNA expression of various lysosomal enzymes cultured with or without nifedipine in gingival fibroblasts. The expressions of mRNA encoding various lysosomal enzymes were examined in gingival fibroblasts cultured with (nifedipine) or without (−) 100 ng/ml of nifedipine for the indicated time periods. Total RNA was isolated and was subjected to RT-PCR analysis as described in Materials and Methods. Values are representative of the results obtained from several gingival fibroblasts from different donors, since all cells tested showed similar results. Expression of cathepsin-L mRNA was markedly suppressed in gingival fibroblasts after 1 day of incubation with nifedipine, while that of other lysosomal enzymes was not apparently influenced. HeLa cells served as the positive control.

Article Snippet: Reagents The blocker of L-type Ca (2+) channels, nifedipine, was from Bayer AG Pharma Research Center (Wuppertal, Germany).

Techniques: Expressing, Cell Culture, Isolation, Reverse Transcription Polymerase Chain Reaction, Incubation, Positive Control

Journal:

Article Title: Cathepsin-L, a Key Molecule in the Pathogenesis of Drug-Induced and I-Cell Disease-Mediated Gingival Overgrowth

doi:

Figure Lengend Snippet: The effects of TG on the activity of cathepsins. Gingival fibroblasts were cultured with or without 100 ng/ml of nifedipine in the presence or absence of the indicated concentrations of Ca mobilizer TG. Twenty-four hours later, cellular proteins were extracted and were subjected to enzyme assay. TG suppressed both cathepsin-(B+L) and -B activity, while nifedipine alone specifically suppressed cathepsin-(B+L) activity. Values are expressed as the % activity against that of controls (day 0).

Article Snippet: Reagents The blocker of L-type Ca (2+) channels, nifedipine, was from Bayer AG Pharma Research Center (Wuppertal, Germany).

Techniques: Activity Assay, Cell Culture, Enzymatic Assay