Journal: bioRxiv

Article Title: A Compendium of Kinetic Cell Death Modulatory Profiles Identifies Ferroptosis Regulators

doi: 10.1101/826925

Figure Lengend Snippet: a , A sub-cluster of mTOR and dual mTOR/PI3K inhibitors, from the larger compendium (purple bar in ), that block ferroptosis induced by system x c - inhibition (sx c - i). b , Cell death over time measured using STACK in three cell lines. INK128 and AZD8055 were used at 1 µM. c , Immunoblotting for phosphorylation and total levels of the canonical mTOR target, 4EBP1. d , Lipid ROS detected using C11 BODIPY 581/591. U-2 OS cells were treated for 22 h then lipid ROS were imaged using C11 BODIPY 581/591. Erastin2 was used at 2 µM. Fer-1: ferrostatin-1. Scale bar = 20 µm. Imaging was performed three times and representative images from one experiment are shown. e , Relative concentration of reduced (GSH) and oxidized (GSSG) glutathione in HT-1080 cells detected using liquid chromatography coupled to mass spectrometry (LC-MS). Erastin was used at 10 µM and INK128 at 5 µM. *P < 0.05, one-way ANOVA with Bonferroni post-tests (n = 4). f , Cell death over time in U-2 OS N cells. Compound concentrations were: erastin2 (2 µM), buthionine sulfoximine (BSO, 1 mM), INK128 (1 µM), rapamycin (Rapa., 100 nM). g , Fold-change amino acids levels in HT-1080 cells determined using LC-MS. INK128 was used at 5 µM and rapamycin at 100 nM. h , Correlations between gene expression and sensitivity to ferroptosis-inducing agents in the CTRP dataset. Genes encoding ribosomal subunits are colored purple. i , Cell death over time in U-2 OS N cells treated ± erastin2 (2 µM) ± cycloheximide (CHX, 10 µg/L) ± BSO (1 mM). Data in b , f and i are mean ± SD from three independent experiments.

Article Snippet: Primary antibodies used were goat anti-actin (I-19, sc-1616, dilution: 1:1000) from Santa Cruz, rabbit anti-RPS6 (2217S, 1:5000), rabbit anti-phospho-RPS6 Ser235/236 (4858S, 1:2000), rabbit anti-4EBP1 (9644S, 1:1000), rabbit anti-phospho-4EBP1 Thr37/46 (9459S, 1:1000), rabbit anti-ATF4 (11815S, 1:1000) and rabbit anti-GAPDH (2118S, 1:500) from Cell Signaling Technologies, rabbit anti-LC3B (NB600-1384AF700, 1:1000) from Novus Biologicals, and mouse anti-tubulin (MS581P1, 1:10,000) from Fisher Scientific.

Techniques: Blocking Assay, Inhibition, Western Blot, Imaging, Concentration Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: bioRxiv

Article Title: A Compendium of Kinetic Cell Death Modulatory Profiles Identifies Ferroptosis Regulators

doi: 10.1101/826925

Figure Lengend Snippet: a , Cell death in medium containing dialyzed serum and formulated to lack arginine (Arg) or leucine (Leu). Erastin2 (Era2) was used at 2 µM. b , Images of cells at 48 h. Scale bar = 25 µm. c , Lipid ROS detected in C11 BODIPY 581/591. U-2 OS cells were treated as indicated for 24 h prior to imaging. Erastin2 was used at 2 µM, ferrostatin-1 (Fer-1) at 1 µM. Non-ox: non-oxidized; Ox: oxidized. Scale bar = 20 µm. Imaging was performed twice and representative images from one experiment are shown. d , Cell death at 48 h. Compound concentrations were: erastin2 (2 µM), BSO (1 mM). e , Immunoblotting for the mTOR readouts phospho-RPS6 (p-RPS6) and phospho-4EBP1/2 (p-4EBP1/2). INK128 was used at 1 µM. f , Model of the transsulfuration pathway. PPG: propargylglycine. f , Western blot of ATF4 protein levels ± Arg-containing medium ± the GCN2 inhibitor GCN2iB (1 µM). g , Expression of the transsulfuration pathway genes CBS and CTH under the same conditions as in f . h , Cell death in U-2 OS N cells at 48 h ± Arg-containing medium ± erastin2 (Era2, 2 µM) ± the GCN2 inhibitor GCN2iB (1 µM). i , Cell death ± erastin2 (Era2, 2 µM) in medium lacking either or both Arg and lysine (Lys). j,i , Cell proliferation as determined from the ratio of live cell (mKate2 + ) counts at 48 h versus 0 h, in different cell lines and medias ± Arg, Lys or Leu. Data in a , d and g - k are mean ± SD from three independent experiments.

Article Snippet: Primary antibodies used were goat anti-actin (I-19, sc-1616, dilution: 1:1000) from Santa Cruz, rabbit anti-RPS6 (2217S, 1:5000), rabbit anti-phospho-RPS6 Ser235/236 (4858S, 1:2000), rabbit anti-4EBP1 (9644S, 1:1000), rabbit anti-phospho-4EBP1 Thr37/46 (9459S, 1:1000), rabbit anti-ATF4 (11815S, 1:1000) and rabbit anti-GAPDH (2118S, 1:500) from Cell Signaling Technologies, rabbit anti-LC3B (NB600-1384AF700, 1:1000) from Novus Biologicals, and mouse anti-tubulin (MS581P1, 1:10,000) from Fisher Scientific.

Techniques: Imaging, Western Blot, Expressing

Journal: PLoS Neglected Tropical Diseases

Article Title: SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties

doi: 10.1371/journal.pntd.0006446

Figure Lengend Snippet: (A) Human serum albumin (HSA), hemoglobin (Hb), immunoglobulin G (IgG), collagen type I (Col I), fibronectin (Fbn) and high molecular weight kininogen (HMWK) were incubated for 16 h at pH 8.0 in the presence (+) or absence (-) of rSmSP2. The reaction mixtures were subjected to SDS-PAGE and protein stained. (B) HMWK was incubated with rSmSP2 and the reaction mixture was subjected to LC-MS/MS analysis to identify bradykinin peptide released from HMWK. (C) Peptide hormones were incubated with rSmSP2 or with live adults maintained in culture and the cleavage positions (full triangles for rSmSP2, open triangles for adult schistosomes) were identified by mass spectrometry. Residues at the P1 position are in bold and the disulfide connectivity of vasopressin is indicated. (D) Human plasminogen (PLG) was incubated in the presence or absence of rSmSP2 and the reaction mixture was analyzed at different time points. Plasmin proteolytic activity generated during plasminogen processing by rSmSP2 was determined in a kinetic assay with Boc-V-L-K-AMC. Mean values of triplicates are expressed relative to the maximum value (100%). The S.D. values of three replicates are within 10% of the mean. All experiments were performed at least twice with similar results. (E) The processed forms were resolved by SDS-PAGE and visualized by protein staining. The positions for PLG, and plasmin (PL) heavy and light chains are indicated. (F) Human tissue plasminogen activator (tPA) was incubated for 16 h at pH 8.0 in the presence/absence of rSmSP2 and analyzed by SDS-PAGE with protein staining; proteolytic activity generated during tPA processing was monitored in a kinetic assay using Z-G-G-R-AMC. Mean values ± s.d. of triplicates are expressed relative to the maximum value (100%). Two chain tPA is indicated with an arrow.

Article Snippet: Recombinant SmSP2 expressed in P . pastoris (300 ng) was incubated at 37°C with 1–20 μg of human plasminogen (hPLG, R&D Systems), high molecular weight human plasma kininogen (HMWK; Merck), human tissue plasminogen activator (tPA), human serum albumin (HSA), human hemoglobin, calf collagen type I, human fibronectin and rabbit immunoglobulin G (all Sigma-Aldrich) in 25 μL 100 mM Tris-HCl, pH 8.0.

Techniques: High Molecular Weight, Incubation, SDS Page, Staining, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Activity Assay, Generated, Kinetic Assay

Journal: PLoS Neglected Tropical Diseases

Article Title: SmSP2: A serine protease secreted by the blood fluke pathogen Schistosoma mansoni with anti-hemostatic properties

doi: 10.1371/journal.pntd.0006446

Figure Lengend Snippet: SmSP2, secreted from adult schistosomes or localized at the surface, stimulates the degradation of blood clots (thrombolysis panel) by (i) activation of two critical components of the fibrinolytic system, tissue plasminogen activator (tPA) and plasminogen, and (ii) direct degradation of the blood-clot component, fibronectin. SmSP2 modulates vascular tone (vasoregulation panel) by processing bioactive peptide hormones. (i) It releases the vasodilatory bradykinin from kininogen (HMWK) and (ii) degrades the vasoconstrictory peptide, vasopressin. Bradykinin may stimulate the release of tPA from vascular endothelial cells (dashed line) which would promote fibrinolysis. SmSP2 may be regulated by plasminogen activator inhibitor-1 (PAI-1) that inhibits SmSP2 .

Article Snippet: Recombinant SmSP2 expressed in P . pastoris (300 ng) was incubated at 37°C with 1–20 μg of human plasminogen (hPLG, R&D Systems), high molecular weight human plasma kininogen (HMWK; Merck), human tissue plasminogen activator (tPA), human serum albumin (HSA), human hemoglobin, calf collagen type I, human fibronectin and rabbit immunoglobulin G (all Sigma-Aldrich) in 25 μL 100 mM Tris-HCl, pH 8.0.

Techniques: Activation Assay

Journal: mAbs

Article Title: Evaluation of the structural, physicochemical, and biological characteristics of SB4, a biosimilar of etanercept

doi: 10.1080/19420862.2016.1193659

Figure Lengend Snippet: Summarized attributes and analytical conclusions of the biosimilar SB4 in comparison with the reference product (EU-sourced Enbrel®) following extensive similarity exercises.

Article Snippet: The sample was digested with Lys-C (Roche, 11047825001) or trypsin (Roche, 11047841001) at 37°C for 16 hours and subsequently with PNGase F (NEB, P0704L) and sialidase A (Prozyme, GK80040) at 37°C for 4 hours.

Techniques: Molecular Weight, Sequencing, Fluorescence, Chromatography, Labeling, Hydrophilic Interaction Liquid Chromatography, Mass Spectrometry, Spectroscopy, Imaging, Electrophoresis, Size-exclusion Chromatography, Sedimentation, Hydrophobic Interaction Chromatography, Binding Assay, SPR Assay, Neutralization, Activity Assay