Journal: PLOS Pathogens

Article Title: LL-37 selectively targets Plasmodium -infected erythrocytes and exhibits antimalarial activity

doi: 10.1371/journal.ppat.1014062

Figure Lengend Snippet: (a) Comparative hemolysis of uninfected erythrocytes (uRBC) and schizont-infected erythrocytes (iRBC) exposed to LL-37 at its IC 50 and 10 × IC 50 concentrations. (b) Stage-specific hemolysis of P. falciparum -infected erythrocytes treated with LL-37 (IC 50 ). R: Ring stage; T: Trophozoite stage; S: Schizont stage. (c) Hemolysis of LL-37 under increasing A23187 concentrations with or without 1.5 mM MβCD. (d) Hemolysis of LL-37 under increasing concentrations of MβCD with or without 0.75 μM calcium ionophore A23187. (e) Parasite parasitemia under treatment with different proportions of MβCD-cholesterol complexes with or without LL-37 present. (f) Parasite parasitemia under treatment with different concentrations of DOTAP with or without LL-37 present. (g) Parasite parasitemia of LL-37 treatment with MβCD-cholesterol complexes and/or DOTAP. All data are presented as means ± SEM from three biological replicates. ns: not significant; * p < 0.05, * p < 0.01, *** p < 0.001 indicate statistically significant differences.

Article Snippet: PS externalization was induced by incubating erythrocytes with calcium ionophore A23187 (MCE, HY-N6687) (0.125, 0.25, 0.5, 0.75 and 1 μM) in PBS containing 1 μM CaCl2 for 3 h at 37°C [ ].

Techniques: Infection

Journal: Aging Cell

Article Title: The mitochondrial ATP synthase is a shared drug target for aging and dementia

doi: 10.1111/acel.12715

Figure Lengend Snippet: J147 modulates resting Ca 2+ homeostasis to activate the AMPK / mTOR axis. (a) J147 increases the levels of cytosolic Ca 2+ in HT 22 cells. A23187 was used as a positive control (* p = .0162, *** p = .0002, *** p < .0001, ANOVA ). (b) The Cam KK 2 inhibitor STO ‐609 attenuated J147‐induced activation (100 n m ) of AMPK targets in rat primary cortical neurons. Phosphorylation targets include AMPK (α‐Thr172), Raptor (Ser792), ACC 1 (Ser79), and S6 (Ser235/236); corresponding quantifications for each are shown below. (c) J147 protection against glutamate (2.5 m m ) is reduced in AMPK KO fibroblasts demonstrating a direct role for AMPK in the protective effects of J147 (**** p < . 0001 (10 n m J147),**** p < . 0001 (25 n m J147),*** p < .0001 (100 n m J147))

Article Snippet: For calcium ionophore experiments, A23187 (Tocris 1234) and ionomycin (Cayman 10004974) were added at the indicated concentrations along with J147 for 1 hr.

Techniques: Positive Control, Activation Assay, Phospho-proteomics

Journal: Diabetes

Article Title: AMP-activated protein kinase activation by adrenoceptors in L6 skeletal muscle cells: mediation by alpha1-adrenoceptors causing glucose uptake.

doi: 10.2337/diabetes.55.03.06.db05-0901

Figure Lengend Snippet: FIG. 6. Representative blots of the effect of TPA (1 mol/l, n 4) and A23187 (1 mol/l, n 4) on AMPK phosphorylation in L6 muscle cells. The histograms are means SE of four experiments performed in duplicate.

Article Snippet: Drugs and reagents were purchased as follows: rosiglitazone (Alexis Biochemicals, Lausen, Switzerland); 2-deoxy-[3H]-D-glucose (12 Ci/mmol 1; Amersham Biosciences, Buckinghamshire, U.K.); Gö6976 and Gö6783 (CalBiochem, La Jolla, CA); 2,4,-dinitrophenol (Merck Schucharat OHG, Hohenbrunn, Germany); insulin (Actrapid) (Novo Nordisk, Bagsvaerd, Denmark); [ -32P]ATP (3,000 Ci/mmol) (PerkinElmer Sverige, Upplands Väsby, Sweden); A23187, cirazoline, forskolin, ( )-isoprenaline, LY294002, ( )-norepinephrine, phenylephrine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) (Sigma Chemical, St. Louis, MO); AICAR (Toronto Research Chemicals, North York, Ontario, Canada); and SAMS peptide (Upstate Biotechnology).

Techniques: Phospho-proteomics

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: In vitro characterization of CG LLPS. (A) Scheme used for purifying GFP and 6x-His-tagged CGA or CGB, respectively. Stable lines expressing CGB-GFP and CGA-GFP under a doxycycline-inducible promoter are treated with doxycycline and the calcium ionophore A23187 to induce secretion of the respective proteins in serum-free medium, which is then used for purification using Ni-NTA affinity columns. (B) Plot of CGA generated using PONDR depicting disordered regions in the proteins. Almost 90% of CGA is disordered when analyzed using the VL-XT algorithm. (C) Coomassie-stained gel depicting purified CGA-GFP. Images showing different droplets of CGA-GFP at different protein concentrations at pH 6.1 in presence of 5% PEG 8000. Note that the size of the condensates decreases with decreasing protein concentration. (D) Representative images of solutions containing CGA-GFP buffered at either pH 6.1(left) or pH 7.3 (right). Droplet formation occurs at pH 6.1 and not at pH 7.3. Droplets of CGA-GFP were induced at 4 µM protein concentration in presence of 5% dextran. (E) Images obtained from plating a solution of CGB-GFP (2.5 µM final concentration) to monitor the presence or absence on liquid-like condensates either without or with 250 µM or 5 mM calcium. Note that droplet formation is induced only in the presence of 5 mM calcium. (F) Phase diagram obtained by varying calcium and CGB concentrations after plating solutions on imaging dishes and observing after 15–20 min. Red circles indicate conditions where no droplets were seen, and green circles indicate conditions which favored presence of CGB droplets. (G) Images obtained from plating a solution of CGA-GFP (2.5 µM final concentration) in presence of either 250 µM, 20 or 40 mM calcium to test for the presence or absence of droplet formation. No droplets are seen even at 40 mM which is the highest calcium concentration. (H) Representative images of CGB-GFP (2.5 µM) in presence of different concentrations of zinc. At high concentrations zinc induces formation of insoluble aggregates but at low concentration, it induces CGB-GFP droplets. Source data are available for this figure: .

Article Snippet: Fully confluent cells were induced for protein expression by the addition of doxycycline monohydrate (1 μg/ml; LKT Laboratories) in serum-free DMEM high glucose also containing proteinase inhibitor aprotinin (1 μg/ml; Sigma-Aldrich) and antibiotic A23187 (1 μg/ml; Thermo Fisher Scientific Alfa Aesar) for 15–20 h. Medium was collected and pre-cleared of cells by centrifugation and then by filtration using a 0.45 μm filter.

Techniques: In Vitro, Expressing, Purification, Generated, Staining, Protein Concentration, Concentration Assay, Imaging