Journal: Neuron

Article Title: An Afferent Neuropeptide System Transmits Mechanosensory Signals Triggering Sensitization and Arousal in C. elegans

doi: 10.1016/j.neuron.2018.08.003

Figure Lengend Snippet:

Article Snippet: The CHO-K1 cell line (PerkinElmer, ES-000-A2) was used for receptor activation assays.

Techniques: Virus, Recombinant

Journal: Brain

Article Title: Efficacy of MEDI0618, a pH-dependent monoclonal antibody targeting PAR2, in preclinical models of migraine

doi: 10.1093/brain/awae344

Figure Lengend Snippet: Specificity and potency of anti-PAR2 monoclonal antibody MEDI0618 . ( A ) Live staining of PAR2-expressing (1321N1-hPAR2.cl8) or non-expressing (1321N1 parental) cell lines with MEDI0618 directly conjugated to Alexa Fluor 647. Scale bar = 20 µm. ( B ) Flow cytometry of hPAR2 overexpressing cells (1321N1-hPAR2.cl8) or non-expressing cells (1321N1 parental cell line) or A549 cells endogenously expressing hPAR2 live labelled with MEDI0618. ( C and D ) Calcium imaging from 1321N1-hPAR2 cells pretreated with MEDI0618 hIgG or an isotype control protein. ( C ) Exemplar raw calcium trace from a single well pretreated with MEDI0618 or isotype control antibody both at 1 nM, followed by PAR2 agonist stimulation with matriptase (10 nM). ( D ) Antibody titration of the anti-PAR2 antibodies MEDI0618 or PAR650097 or an isotype control antibody. Data show the matriptase (10 nM)-mediated calcium signal after preincubation with antibody and normalized to the matriptase response in the absence of antibody treatment. ( E ) Calcium imaging from A549 cells pretreated with MEDI0618, isotype control protein or PAR1 inhibitors (ATAP2 + WEDE15 mAbs) followed by 10 nM thrombin (PAR1 agonist) stimulation. Data are presented as mean ± standard error of the mean, n = 4. The concentration of the inhibitor is shown on the x -axis. Data are normalized to the peak thrombin calcium response in the absence of inhibitor pretreatment.

Article Snippet: The 1321N1 parental cell line (lacking PAR2 expression), 1321N1 cells stably expressing human PAR2 (1321N1-hPAR2.cl8) (generated in-house at AstraZeneca) or A549 cells endogenously expressing hPAR2 were pre-stained with LIVE/DEADTM dead cell stain (ThermoFisher); 7.5 × 10 6 cells from each line were collected in total and stained in 7.5 ml phosphate buffered saline (PBS) (ice-cold) with 1× violet dead cell stain for 30 min on ice.

Techniques: Staining, Expressing, Flow Cytometry, Imaging, Control, Titration, Concentration Assay

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Acquired resistance upon long-term 4-HPR exposure . (A, left) R cell lines were continuously exposed to different 4-HPR concentrations (0.5, 3, 5, and 10 μM; cell lines named R0.5, R3, R5, and R10, respectively) and resistance verified by comparison to 48 h drug-withdrawn R cells. Data are the average ± SD of three independent experiments performed in quadruplicates, n = 12. (A, right) Parental CCRF-CEM cells and developed R cell lines were exposed to 10 μM 4-HPR and viability estimated after 48 h of drug exposure. (n = 12). ** P < 0.01; ANOVA plus Tamhane post-hoc analysis. (B) Comparative cell proliferation was determined by BrdU labeling as specified in Materials and Methods. Data are average ± SD of four independent experiments. * P < 0.05; Kruskal Wallis and Mann-Whitney test for pair-wise comparison.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Comparison, Labeling, MANN-WHITNEY

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Effect of selected insults on the viability of 4-HPR-sensitive and resistant leukemia cell lines . 4-HPR-sensitive (CCRF-CEM) and derived resistant cell lines (R0.5 and R10) were exposed to several drugs or UV radiation, and cytotoxicity tested after 48 h by the XTT assay. Data are average ± SD of three independent experiments performed in quadruplicates (n = 12); * P < 0.05, ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Derivative Assay, XTT Assay

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative endogenous SL pattern in 4-HPR-sensitive and resistant leukemia cell lines . Cell were grown with (R cell lines) or without (CCRF-CEM) 4-HPR. Endogenous TOTAL (sum of the different fatty acid chain-length species) dhCer (A, left panel) and Cer (A, right panel) levels, as well as dhSph (B), TOTAL GluCer, and LactCer (C) levels were determined by LC/MS. Data are average ± SD of four (A, B) or two (C) independent experiments. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane or Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative analysis of dihydroceramide desaturase (DES) activity . Cells were incubated with the unnatural pyridinium dhCer (C12-PyrdhCer) alone or with 4-HPR (5 μM) for the selected time points and conversion into C12-PyrCer measured by LC/MS. Data are average ± SD of two independent values. ** P < 0.01; ANOVA plus Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Activity Assay, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Comparative SL profile analysis upon drug withdrawal . R10 cells were incubated in 4-HPR-free medium for 48 h (WD = without drug) or long-term (~3 weeks) and endogenous SL profiles analyzed by LC-MS. Significance was based on R10 values. Data are average ± SD of 2-4 independent values. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane or Bonferroni post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Effect of drug withdrawal on cell resistance . (A) Parental sensitive CCRF-CEM cells, resistant R10, and long-term drug withdrawn resistant (R10 longWD) cells were treated and cell death determined by annexin V-FITC/propidium iodide staining at the selected time points. Values represent the percentage of cells in each quadrant. Representative data from two independent experiments are shown. (B) A portion of each sample for SL profile analysis (Figure 5) was saved for XTT assay estimation of resistance. Briefly, R10 cells after 48 h and long-term drug withdrawal were subjected to 48 h treatment with 3 or 10 μM 4-HPR. All data were related to the corresponding untreated cells. Statistical analysis was related to R10 data. Data are average ± SD of 12 values from to three independent experiments. * P < 0.05; ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Staining, XTT Assay

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Analysis of 4-HPR-mediated alterations in dhSph and Sph phosphorylation . Externally added unnatural dhSph (17C-dhSph) and Sph (17C-Sph) analogues were used to estimate the secondary effects of 4-HPR-induced DES inhibition. CCRF-CEM cells were exposed to 4-HPR (10 μM) for 1 h and unnatural 17C species added for an additional 30 min incubation, as described in Methods. The effect on endogenous SLs (A) and unnatural dhSph species (B,C) analyzed by LC/MS. Comparative exogenous and endogenous SL profiles among parental (CCRF-CEM) and resistant cell lines (R0.5, R10) (D, E) were analyzed by the same methodology. Data are average ± SD of an experiment performed in duplicate.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Phospho-proteomics, Analogues, Inhibition, Incubation, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Cancer

Article Title: Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells

doi: 10.1186/1471-2407-11-477

Figure Lengend Snippet: Toxicity mediated by the combination of 4-HPR with SL modulators . Parental-sensitive (CCRF-CEM) and derived-resistant (R0.5, R5, R10) cells were co-treated with 4-HPR (1 μM), DHS (5 μM; unnatural dhSph analogue), PPMP (10 μM; glucosylceramide synthase inhibitor), SAF (4 μM; another unnatural dhSph analogue), and/or SKI-II (5-20 μM) and cytotoxicity estimated after 48 h exposure using the XTT assay. Data are average ± SD of at least two independent experiments performed in quadruplicate (n ≥ 8); ** P < 0.01; ANOVA plus Tamhane post-hoc test.

Article Snippet: 4-HPR-sensitive parental leukemia cell line (CCRF-CEM) and 4-HPR-resistant cell lines (R0.5 and R10) were challenged by different anticancer drugs, such as cisplatin (0.5, 1, 2.5, or 5 μg/ml), paclitaxel (1, 5, 10, 30, or 100 nM), or adriamycin (50, 100, or 500 nM) and other insults such as UV irradiation (1, 3, or 5 mJ/cm ) or direct oxidative stress (H O 2; 10, 50, or 100 μM).

Techniques: Derivative Assay, XTT Assay