Journal: Molecular Oncology

Article Title: UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

doi: 10.1002/1878-0261.12372

Figure Lengend Snippet: LoVo cells treated with VTD show a dose‐dependent reduction in mot‐2 levels. (A, B) LoVo colon cancer cells were treated with the indicated concentrations of VTD or vehicle (DMSO) for 72 h followed by staining with anti‐mot‐2/Alexa Fluor 546. Cells were examined using flow cytometry, and data were normalized with DMSO. Data are shown as mean ± SEM of three data readings ( n = 3) where * P < 0.05 and ** P < 0.01 using Tukey's modified Student's t ‐test. (C, D) In a similar set of experiments, LoVo cells were treated with DMSO or different concentrations of VTD for 72 h. Cell lysates were subjected to WB experiments. Statistical analysis of mot‐2 protein bands normalized by β‐actin shows VTD significantly deceases mot‐2 proteins in a dose‐dependent manner ( n = 3, * P < 0.05, ** P < 0.01, *** P < 0.001 Tukey's modified student's t ‐test, mean ± SEM). (E) Three groups of LoVo cells were treated with DMSO, VTD (30 μ m ), or VTD (30 μ m ) plus bortezomib for 72 h, followed by cell lysate preparation. Bortezomib was added 24 h before preparing cell lysates. WB analysis showed VTD‐dependent reduction in mot‐2 can be reversed by bortezomib, while VTD showed no effects on HSC70. Lower Panel E shows the efficient accumulation of ubiquitinated proteins in the presence of bortezomib. Experiments in Panel E were repeated two times with similar results.

Article Snippet: Veratridine (VTD), an alkaloid extracted from the Veratrum officinale plant, was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Staining, Flow Cytometry, Modification, Analysis, FACS

Journal: Molecular Oncology

Article Title: UBXN2A enhances CHIP‐mediated proteasomal degradation of oncoprotein mortalin‐2 in cancer cells

doi: 10.1002/1878-0261.12372

Figure Lengend Snippet: VTD selectively decreases protein levels of mot‐2 and its chaperone partner, HSP60, in both cytoplasmic and mitochondrial compartments. LoVo cells were treated with 100 μ m VTD for 72 h followed by iodixanol gradient ultracentrifuge fractionations. (A–C) ER, Golgi, and mitochondrial markers determined the locations of these three membrane compartments. (D) Collected fractions (1–18) were probed with anti‐mot‐2, anti‐HSP60, and anti‐HSC70 antibodies. Results show a dramatic reduction in mot‐2 and HSP60 in both cytosolic‐ and mitochondrial‐enriched fractions, whereas HSC70 levels slightly decreased in the presence of VTD.

Article Snippet: Veratridine (VTD), an alkaloid extracted from the Veratrum officinale plant, was purchased from Alomone Labs (Jerusalem, Israel).

Techniques: Membrane, Western Blot, Enzyme Activity Assay

Journal: SLAS discovery : advancing life sciences R & D

Article Title: Three-Dimensional Control of Ion Channel Function through Optogenetics and Co-Culture.

doi: 10.1177/2472555217722990

Figure Lengend Snippet: Figure 3. Modulation of ligand- or light-induced stimulation of voltage-gated sodium channel by Kir2.3 co-culture. (A) Syncytial populations between a fixed amount (3000 c/w) of a voltage-gated sodium channel (VGSC) cell line and different amounts (10, 50, 100, 200, 500, 1000, 2000 c/w) of a Kir2.3 inward-rectifier potassium channel cell line were stimulated by veratridine application (50, 100, or 150 µM) and simultaneously imaged by a fluorescent membrane potential sensitive organic Probe (FMP) in Tyrode’s buffer. Monoculture assay of VGSC alone was analyzed in parallel, as internal reference control. Stimulation of VGSC with veratridine opener was significantly enhanced by co-culture with moderate amounts of Kir2.3 cell line, either when using 50, 100, or 150 µM concentration. The maximal potentiating effect was obtained with a 15:1 ratio of VGSC (3000 cells/well) versus Kir2.3 cells (200 cells/well). In the absence of Kir2.3 cells or in the presence of high amounts of Kir2.3 cells, veratridine opener elicited smaller VGSC stimulation. (B) Syncytial population between a ChR2-VGSC (3000 cells/well) and a Kir2.3 (200 cells/well) cell line was stimulated with FLIPR Tetra Blue light-emitting diodes (100 ms duration, 3.5 Hz) and simultaneously imaged with an FMP in K0Na150 buffer plus increasing concentrations of amitriptyline VGSC blocker. Monoculture assay of ChR2-VGSC alone was analyzed in parallel, as internal reference control. In the absence of Kir2.3 cells (dashed line), only the amitriptyline- insensitive ChR2-mediated depolarization was detected, with modest signal amplitude because of the depolarized membrane voltage. In the presence of the optimal Kir2.3 cell number (200 cells/well), light depolarization showed an amitriptyline-sensitive component with a pharmacology compatible with resting block of VGSC and a residual amitriptyline-insensitive component mediated by ChR2, with a significant signal amplitude due to the hyperpolarized membrane voltage.

Article Snippet: All-trans Retinal, amitriptyline hydrochloride, carbachol, and DMSO were from Sigma Aldrich (St. Louis, MO); potassium chloride was from DelChimica (Italy); veratridine and isradipine were from TOCRIS Bioscience (Bristol, UK); and selective antibiotics were from Invivogen (Thermo Scientific, Waltham, MA).

Techniques: Co-Culture Assay, Membrane, Control, Concentration Assay, Blocking Assay

Journal: Marine Drugs

Article Title: Identification of New CTX Analogues in Fish from the Madeira and Selvagens Archipelagos by Neuro-2a CBA and LC-HRMS

doi: 10.3390/md20040236

Figure Lengend Snippet: Dose-responses curves of neuro-2a cells to CTX1B or a liver fish extract, in the absence of ouabain and veratridine (O/V−) and the presence of ouabain and veratridine conditions (O/V+): ( a ) Dose-response curve of neuro-2a cells after the exposition to CTX1B standard; ( b ) Dose-response curve of neuro-2a cells after the exposure to liver fish extract (specimen with code 35). Data represent the mean and SD of three replicates on the same day of experimentation.

Article Snippet: Poly-L-lysine, foetal bovine serum (FBS), L-glutamine solution, ouabain (O), veratridine (V), phosphate buffered saline (PBS), penicillin, streptomycin, RPMI-1640 medium, sodium pyruvate, thiazolyl blue tetrazolium bromide (MTT) were purchased from Merck KGaA (Darmstadt, Germany).

Techniques: