Journal: Oncotarget

Article Title: PTEN mediates the cross talk between breast and glial cells in brain metastases leading to rapid disease progression.

doi: 10.18632/oncotarget.14047

Figure Lengend Snippet: Figure 7: Effects of glial cell cocultures on cytokine secretion. Control and PTEN overexpressing MDA-MB-231 BR cells were seeded in transwell inserts and cocultured for 48 h with astrocytes (A) or microglia (B). Antibody-spotted membranes were incubated with conditioned media of these cultures and levels of secreted proteins were visualized by HRP-conjugated antibodies. Proteins with a more than 2-fold expression difference are shown by open boxes. Results were verified by qPCR analyses (C). Astrocyte coculture reduced EGF and GM-CSF expression significantly in PTEN overexpressing (PTEN CoA) compared to control cells (CTL CoA, C). Astrocytes express the GM- CSF receptor (CSFR2A) only when cocultured with tumor cells. Microglia coculture reduced VEGFA expression in PTEN overexpressing (PTEN CoMG) compared to control cells (CTL CoMG). Student’s t-test was used to analyze statistical significance. *p < 0.05.

Article Snippet: Cytokine secretion was estimated from CM by Proteome ProfilerTM Antibody Array (Human XL Cytokine Array Kit, ARY022, R&D Systems, Wiesbaden, Germany) according to manufacturer’s instruction.

Techniques: Control, Incubation, Expressing

Journal: Acta Oncologica

Article Title: Identification of a novel resistance mechanism in venetoclax treatment and its prediction in chronic lymphocytic leukemia

doi: 10.1080/0284186x.2021.1878388

Figure Lengend Snippet: Figure 2. Change of protein expression during venetoclax treatment measured by apoptosis arrays.

Article Snippet: Apoptosis array (R&D Systems Minneapolis, USA, Human apoptosis antibody array kit) was performed from the mononuclear cell fraction of the samples according to the manufacturer’s instructions (Analysis ImageJ software version 1.50d, NIH, USA) [10,11].

Techniques: Expressing

Journal: Microbiology Spectrum

Article Title: Density Analysis of Enterovirus D68 Shows Viral Particles Can Associate with Exosomes

doi: 10.1128/spectrum.02452-21

Figure Lengend Snippet: Immune recognition of various EV-D68 densities and characterization of membrane-associated virus. (A) The y axis represents average dilutions of anti-EV-D68 mouse serum required to neutralize virus, divided by the average TCID 50 for respective viral densities (ANOVA post hoc Student'’s t test, P = 0.42, P = 0.68, P = 0.70). (B) Three viral density isolates (1.11, 1.20, and 1.24 g/cm 3 ) were treated with 0.01 mg/mL 15C5-Chmra antibody for 1 h, then mix was put onto TCID 50 plates to assess the viral titer of each isolate. Gray highlight represents detection limit. Asterisks (*) indicate statistical significance (1.11 g/cm 3 , P = 0.0003; 1.20 g/cm 3 , P < 0.0001; 1.24 g/cm 3 , P = 0.0064), all versus respective control, determined by Dunnett’s Method. (C) 15C5-Chmra antibody bound to magnetic beads was added to membrane-associated and naked virus. After 1 h, a magnet was used to remove antibody and the supernatant was added to a TCID 50 plate to assess viral titer (15C5-Chmra versus control: *, P < 0.0005 for both membrane-associated and naked virus; Dunnett’s Method). (D) ICAM-5 or N -acetylneuraminic acid (sialic acid) were attached to magnetic beads and the antibody/bead complex was incubated with membrane-associated or naked virus samples for 1 h. Beads were rinsed twice in excess PBS and viral titer was assessed to determine how much virus was immunoprecipitated from the supernatant (control versus ICAM5 and control versus sialic acid for membrane-associated and naked virus; *, P = 0.0001 determined by Dunnett’s Method). (E) Exosome antibody array on 1.11 g/cm 3 fraction, examining cytosolic proteins (FLOT1, ALIX, TSG101), transmembrane proteins (CD63, CD81, ANXA5), and cis -golgi matrix protein as markers for cellular contamination (GM130). Example blot is shown on the right and chart represents average intensity across three biological replicates. Positive control indicates detection reagents are working correctly, and do not represent an exosome-specific control. Error bars represent standard deviation. Statistics: comparison with control (blank) using Dunnett’s Method ( P = 0.999 for GM130; *, P = 0.027 for FLOT1; P = 0.218 for ICAM; *, P = 0.005 for ALIX; P = 0.086 for CD81; *, P < 0.0001 for CD63; P = 0.305 for EpCAM; *, P < 0.0001 for ANXA5; *, P = 0.0008 for TSG101). Asterisks indicate statistical significance. (F) Anti-CD81 or anti-CD63 antibodies were attached to magnetic beads and incubated with membrane-associated virus. Supernatant was discarded, and beads were rinsed and treated with 0.01% NP-40 (to dissolve exosomes and release virus from bead) before TCID 50 measurement. CD81 versus control: *, P = 0.0178; CD63 versus control: *, P = 0.0180 as determined by Dunnett’s Method. Gray highlight represents detection limit. (G) RD or SH-SY5Y cells in TCID 50 plate were infected with MO47 with or without exosomes in the medium. The “A549 exosomes added” bar represents exosome-depleted media to which purified A549 exosomes were added. Gray panel represents TCID 50 plates containing SH-SY5Y cells. Each condition represents 3 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05. Green panel represents TCID 50 plates containing RD cells. Each condition represents 4 biological replicates. Error bars represent standard deviation. ANOVA: *, P < 0.05.

Article Snippet: We followed the coupling protocol from the Dynabeads Antibody Coupling Kit (Thermo Fisher, cat no. 14311D) to covalently attach magnetic beads to the following antibodies: anti-CD81 (1D6) monoclonal antibody (Novus Biologicals NB100-65805), anti-CD63 (H5C6) monoclonal antibody (Novus Biologicals NBP2-42225), 15C5-chimeric monoclonal antibody (generous gift from Michael Pauly at ZabBio), and anti-HSV negative control (generous gift from Michael Pauly at ZabBio).

Techniques: Membrane, Virus, Control, Magnetic Beads, Incubation, Immunoprecipitation, Ab Array, Positive Control, Standard Deviation, Comparison, Infection, Purification

Journal: Oncology Letters

Article Title: MicroRNA profiles during galectin-9-induced apoptosis of pancreatic cancer cells

doi: 10.3892/ol.2017.7316

Figure Lengend Snippet: CCK18 produced specifically during apoptosis was identified by ELISA. Cells were incubated with or without 0.3 µM Gal-9. Gal-9 increased the levels of CCK18 in PK-1 cells, suggesting that the apoptotic process following phosphatidylserine exposure proceeds to cut the intermediate filaments of cells. Fold-change in CCK18 is shown as the mean ± standard deviation, with analysis by a paired t-test. For each cell line, the condition at 24 h was significantly different from that of the control. CCK18, caspase-cleaved cytokeratin-18; Gal-9, galectin-9.

Article Snippet: A Human Apoptosis Antibody Array kit (R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer's protocol.

Techniques: Produced, Enzyme-linked Immunosorbent Assay, Incubation, Standard Deviation, Control

Journal: Oncology Letters

Article Title: MicroRNA profiles during galectin-9-induced apoptosis of pancreatic cancer cells

doi: 10.3892/ol.2017.7316

Figure Lengend Snippet: The effects of Gal-9 on apoptosis-related proteins in PK-1 cells. (A) Template showing the locations of tyrosine kinase antibodies spotted onto a human apoptosis array. (B) Representative expression of various apoptosis-related proteins in PK-1 cells treated with or without Gal-9. (C) Densitometry analysis demonstrated that the intensity of cytochrome c spots from Gal-9-treated cells relative to untreated cells was 129.7%. Gal-9, galectin-9; Bad, Bcl-2 associated death promotor; Bax, Bcl-2 associated × protein; Bcl-2, B-cell lymphoma 2; Bcl-x, B cell lymphoma-extra-large; clAP, cellular inhibitor of apoptosis protein; Trail R1/DR4, Trail receptor 1/death receptor 4; FADD, Fas associated via death domain; TNFRSF6/CD95, tumor necrosis factor receptor superfamily member 6/cluster of differentiation 95; HIF-1α, hypoxia-inducible factor-1α; HO-1/HMOX1/HSP32, heme oxygenase-1/heme oxygenase (decycling) 1/heat shock protein 32; PON2, paraoxonase 2; P21/CIP1/CDKN1A, cyclin-dependent kinase inhibitor 1A; SMAC, second mitochondria-derived activator of caspases; TNF R1, tumor necrosis factor receptor 1; TNFRSF1A, TNF receptor superfamily member 1A; XIAP, X-linked inhibitor of apoptosis protein.

Article Snippet: A Human Apoptosis Antibody Array kit (R&D Systems, Inc., Minneapolis, MN, USA) was used according to the manufacturer's protocol.

Techniques: Expressing, Derivative Assay

Journal: Communications Biology

Article Title: Macrophage migration inhibitory factor is overproduced through EGR1 in TET2 low resting monocytes

doi: 10.1038/s42003-022-03057-w

Figure Lengend Snippet: a , b Cytokine profile arrays of supernatant collected from cord blood CD34 + cells infected with SCR - and TET2- shRNA-GFP lentiviruses, sorted on GFP expression, and induced to differentiate with stem cell factor (SCF), interleukin-3 (IL-3), Fms-related tyrosine kinase 3 ligand (FLT3L) and granulocyte-colony stimulating factor (G-CSF). a Representative cytokine array with supernatants collected at day 10 of differentiation. The rectangle points to MIF detection. b Quantification of MIF signals, normalized to positive controls. Data are mean +/− SEM of three independent experiments. Paired t test: * P < 0.05. c MIF concentrations determined by ELISA in the supernatant of cells induced to differentiate for indicated time. Data are mean +/− SEM of indicated independent experiments (day 5: n = 6; day 7: n = 5; day 8: n = 3; day 10: n = 7). Paired t test: * P < 0.05; *** P < 0.001. d RT-qPCR analysis of MIF mRNA expression in four TET2 -depleted ( TET2 shRNA, gray bars) and control ( SCR shRNA, black bars) human leukemic cell lines. Data are mean +/− SEM of three biological replicates. Unpaired t test: * P < 0.05; *** P < 0.001; **** P < 0.0001. e Immunoblot of SCR or TET2 shRNA infected leukemic cell lines sorted on GFP expression. Lower panels, quantification after actin normalization using Image J software. f MIF concentrations determined by ELISA in the supernatants of kasumi-1 ( n = 5), M07e ( n = 5), UT-7 ( n = 4) and TF-1 ( n = 5) cells transduced 24 h before with SCR (black squares) or TET2 (gray squares) shRNA. Data are mean +/− SEM of indicated biological replicates. Unpaired-t test: * P < 0.05; ** P < 0.01. g MIF concentrations determined by ELISA in the plasma of two Tet2 -deficient models (1–3 months): K427 knock-out model (wt/wt or Tet2 +/+ n = 4, wt/exc or Tet2 +/− n = 5 and exc/exc or Tet2 −/− n = 5); ANO knock-down model (wt/wt or Tet2 + /+ n = 5, wt/LacZ or Tet2 +/− n = 5 and LacZ/LacZ or Tet2 −/− n = 6). Data are mean +/− SEM of indicated biological replicates. Dunnett’s multiple comparison tests using wt/wt as control: * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: CD34 + collected supernatants were analyzed using human cytokine antibody array (panel A; R&D Systems).

Techniques: Infection, shRNA, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control, Western Blot, Software, Clinical Proteomics, Knock-Out, Knockdown, Comparison