Journal: Cell reports

Article Title: Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response

doi: 10.1016/j.celrep.2024.113882

Figure Lengend Snippet:

Article Snippet: Cercopithecus aethiops: BSC-40 kidney epithelial cells (female) , ATCC , CRL-2761.

Techniques: Virus, Control, Recombinant, Lysis, Staining, Protease Inhibitor, Magnetic Beads, Modification, Saline, Expressing, Transfection, Blocking Assay, Fractionation, HTS Assay, Plasmid Preparation, Software, Imaging

Journal: iScience

Article Title: Activity-dependent COX-2 proteolysis modulates aerobic respiration and proliferation in a prostaglandin-independent manner

doi: 10.1016/j.isci.2024.111403

Figure Lengend Snippet:

Article Snippet: Human HEK 293 passage 13-40 , ATCC , CRL-1573.

Techniques: Recombinant, Protease Inhibitor, Staining, Labeling, Membrane, In Vitro, Transfection, Electrophoresis, Sequencing, Modification, Enzyme-linked Immunosorbent Assay, Mutagenesis, Plasmid Preparation, Software

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Generation and characterization of scR1antTNF-Fc protein. A, schematic structure modeling of scR1antTNF-Fc protein. Two TNFR1 antagonistic proteins were fused with human IgG-Fc. N, N-terminal. Amino acid sequences and domain information of scR1antTNF-Fc are described in Fig. S1A. B, X-ray structure modeling of the scR1antTNF-TNFR1 complex. scR1antTNF bound to the homotrimer of TNFR1. Red, TNFR1; green, TNFR1 interaction domain of scR1antTNF; orange, peptide linker for forming the single-chain structure. C, schematic pCAG-based mammalian expression vector for scR1antTNF-Fc protein. The cDNA was composed to express scR1antTNF-Fc whereby triple R1antTNF domains fused by peptide linkers (GGGSGGG) were further fused to a human–IgG Fc domain (Ch2 and Ch3). Signal sequence peptide genes derived from a mouse IgG Vh (Vhss) or human IL-2 (IL-2ss) were linked at the 5′-terminal of scR1antTNF-Fc cDNA. D, supernatants of cultured medium (left side) and purified proteins (right side) 7 days after transfection were assessed by SDS-PAGE following Coomassie Brilliant Blue staining. Arrowhead shows an ∼75-kDa band of the scR1antTNF-Fc monomer. E, each recombinant protein expressed with Vhss and IL-2ss was purified by size-exclusion chromatography. F, the molecular weight of monomeric scR1antTNF-Fc protein was confirmed by Western blotting with an anti-human IgG-Fc antibody.

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Expressing, Plasmid Preparation, Sequencing, Derivative Assay, Cell Culture, Purification, Transfection, SDS Page, Staining, Recombinant, Size-exclusion Chromatography, Molecular Weight, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: In vitro binding affinity of scR1antTNF-Fc. A, in vitro receptor-binding ability of human TNFα, scR1antTNF, and scR1antTNF-Fcs to human TNFR1 and human TNFR2 was analyzed by SPR. Each sensorgram shows the association (120 s) and dissociation (120 s) repeats at five serial concentrations (1.2, 3.7, 11.1, 33.3, and 100 nm) using single-cycle kinetics. Analytes: TNFα, scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss). Ligands: Fc chimera proteins of human TNFR1 and human TNFR2. B, kinetic parameters of each protein to human TNFR1/TNFR2 were analyzed with a 1:1 binding model using BIAcore × 100 evaluation software (n = 1). The avidity of scR1antTNF-Fc (Vhss) and scR1antTNF-Fc (IL-2ss) were analyzed as a bivalent analyte. Kd, ka, and kd indicated the dissociation constant, association rate constant, and dissociation rate constant, respectively.

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: In Vitro, Binding Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Thermal stability of scR1antTNF-Fc. A, thermal stabilities of scR1antTNF, scR1antTNF-Fc (Vhss), scR1antTNF-Fc (IL-2ss) and etanercept were measured by thermal shift assay using differential scanning fluorometry. Proteins serially diluted from 250 μg/ml by 2-fold dilution are indicated. B, temperature of the peak apex of five concentrations show the denaturation temperature (Tm). Tm values calculated from the result of thermal shift assay using Protein Thermal Shift Software.

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Thermal Shift Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: In vitro bioactivity of scR1antTNF-Fc via TNFR1 or TNFR2. A, in vitro agonistic bioactivities of scR1antTNF or scR1antTNF-Fcs through TNFR1 for LM cells were measured. Human TNFα was used as a control. B, antagonistic activities of scR1antTNF, scR1antTNF-Fc (Vhss), and scR1antTNF-Fc (IL-2ss) via TNFR1 were confirmed by LM cell assay. LM cells were treated with each protein in the presence of mouse TNFα (5 ng/ml). The TNF inhibition rate was determined from the LM cell viability. C, NF-κB induction was evaluated by reporter assay using Ramos-Blue cells. Ligand-dependent SEAP activities were detected. D, agonistic activity via TNFR2 was evaluated by the cell death of huTNFR2/mFas preadipocytes. Data are shown as the mean ± S.D. (n = 3).

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: In Vitro, Control, Inhibition, Reporter Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: In vivo plasma clearance of scR1antTNF-Fc. A, plasma clearances of scR1antTNF, scR1antTNF-Fc, and etanercept were confirmed after i.p. injection. Etanercept was used as a positive control. Plasma concentrations of these proteins were measured by ELISA for human TNF or human IgG-Fc. Data are shown as the mean ± S.D. of five mice per group. B, half-lives and AUCs were calculated from time-concentration curves by moment analysis.

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: In Vivo, Injection, Positive Control, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: scR1antTNF-Fc treatment suppresses inflammation in arthritis mice. A, DBA/1 mice were immunized by the subcutaneous injection of bovine type II collagen with CFA. Saline (n = 6), etanercept (1250 μg/kg) (n = 6), and scR1antTNF-Fc (50 μg/kg) (n = 6) were administered i.p. twice a week from day 22 after immunization. B and C, sum of arthritis scores of four paws (B) and body weight (C) were measured for 3 weeks. D, arthritis incidence was calculated from the number of mice with swollen limbs. E, joint swelling in a representative individual from each group at day 42 is shown. Data are shown as the mean ± S.E.; *p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Injection, Saline

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Effects of scR1antTNF-Fc treatment on joint pathology and blood cytokine levels. A, after treatment of CIA mice with saline, etanercept (1250 μg/kg), or scR1antTNF-Fc (50 μg/kg) for 3 weeks (on day 42), histological sections of the ankle joint from a hind limb were prepared and stained with H&E. B, histopathologic features such as cell infiltration, synovitis, destruction of cartilage, and the juxta-articular bone involvement on day 42 were scored. C, TRAP-positive cells were stained using serial sections to detect osteoclasts. TRAP-positive cells with ≥1 nucleus were counted as indicated by an arrow. D, mouse IL-1β (n = 6) in plasma on day 42 was measured by ELISA. Etanercept, 1250 μg/kg; scR1antTNF-Fc, 50 μg/kg. Data are shown as the mean ± S.D.; *, p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Saline, Staining, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: Effect of scR1antTNF-Fc administration on T cell subpopulations. At day 42, lymph nodes were isolated from CIA mice administered i.p. with saline (n = 6), etanercept (1250 μg/kg) (n = 6), or scR1antTNF-Fc (50 μg/kg) (n = 6) for 3 weeks. After single cells were prepared, the expressions of lymphocyte markers were analyzed by flow cytometry. A, representative flow cytometry data of each administration group are shown. T cells were separated by CD8, CD4, and Foxp3 expression levels. B, the percentage of CD4+ T cells and CD8+ T cells in lymph node cells was analyzed. C, the percentage of CD4+ Foxp3+ Tregs and CD4+ Foxp3− Tconvs in CD4+ T cells was measured. The ratio of Tregs/Tconvs was calculated from the results of individual mice. Data are shown as the mean ± S.D. of eight mice per group; *, p < 0.05, **, p < 0.01 (one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Isolation, Saline, Flow Cytometry, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Structural optimization of a TNFR1-selective antagonistic TNFα mutant to create new-modality TNF-regulating biologics

doi: 10.1074/jbc.RA120.012723

Figure Lengend Snippet: PEGylated scR1antTNF suppresses arthritis in CIA mice as well as scR1antTNF-Fc. A, schematic structure modeling of 40-kDa PEG-scR1antTNF. Branched PEG, which has two 20-kDa PEG chains, was fused to scR1anTNF on an N-terminal amine group. Amino acid sequences, domain information, and molecular modification sites of 40-kDa PEG-scR1antTNF are described in Fig. S1B. B, saline (n = 8), etanercept (1250 μg/kg) (n = 8), scR1antTNF-Fc (10 μg/kg) (n = 8), and 40-kDa PEG-scR1antTNF (10 μg/kg) (n = 8) were administered i.p. twice a week to CIA mice from day 22 after immunization. Arthritis scores were evaluated for 3 weeks. Data are shown as the mean ± S.E.; *, p < 0.05 (one-way ANOVA with Tukey's multiple comparisons test). C, body weight was measured from day 22 to day 35. D, wrist joint swelling of a representative mouse in each group at day 35 is shown. E, the percentages of CD4+ T cells and CD8+ T cells in lymph node cells were analyzed in each treatment group on day 35 by FCM. F, the percentages of CD4+ Foxp3+ Tregs and CD4+ Foxp3− Tconvs in CD4+ T cells were analyzed at day 35. The ratio of Tregs/Tconvs was calculated from the results of individual mice. Data are shown as the mean ± S.D.; *, p < 0.05; **, p < 0.01 (one-way ANOVA with Tukey's multiple comparisons test).

Article Snippet: Induction of arthritis and administration of scR1antTNF-Fc A mixture of bovine collagen type II (2 mg/ml, Chondrex, Redmond, WA, USA) and complete Freund's adjuvant (CFA, 3 mg/ml, Chondrex) were prepared according to a standard collagen-induced arthritis protocol by Chondrex, Inc. Then, 100 μl of the emulsion was subcutaneously immunized in DBA/1 mice (male, 6 weeks old) at the base of the tail. scR1antTNF-Fc, 40-kDa PEG-scR1antTNF, etanercept (positive control) and saline (negative control) were administered i.p. twice a week from day 22 after immunization.

Techniques: Modification, Saline

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on methylglyoxal (MGO)-induced cell toxicity in MES13 cells. ( A ) Chemical structure of ( a ); l -cysteine, ( b ); N -Acetyl- l -cysteine (NAC), ( c ); Cystine. ( B ) Cell viability of MES13 cells treated with MGO (500 μM) and equal concentrations (1.0 mM) of l -cysteine, NAC, and cystine. ( C ) Cell viability of MES13 cells treated with MGO (500 μM) and various concentrations of l -cysteine (0.1, 0.5, and 1.0 mM) and analyzed using the MTT assay. ( D ) Representative photographs of l -cysteine pre-treatment protection against MGO-induced cytotoxicity. Scale bar indicates 500 μm. ( E ) Quantitative measurements of confluence were evaluated using IncuCyte Zoom imaging system. ( F ) MGO-induced LDH production was evaluated by LDH assay in MES13 cells. All data are presented as mean ± SEM. n = 3 (### p < 0.001 vs. Control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: MTT Assay, Imaging, Lactate Dehydrogenase Assay, Control

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on MGO-induced apoptosis and reactive oxygen species (ROS) generation in MES13 cells. ( A ) Representative cytograms of Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining of MGO-induced MES13 cells. Cells were pretreated with several concentrations l -cysteine for 1 h, then incubated with MGO (500 μM) for 24 h. After 24 h, the concentrations of viable (Annexin V-FITC and PI negative cells), early-stage apoptotic (Annexin V-FITC positive, PI negative cells), late-stage apoptotic (Annexin V-FITC positive, PI-positive cells), and necrotic (PI-positive cells) cells were analyzed by flow cytometry. ( a ) control; ( b ) 500 μM MGO; ( c ) MGO+ l -cysteine (0.1 mM); ( d ) MGO+ l -cysteine (0.5 mM); ( e ) MGO+ l -cysteine (1.0 mM); ( f ) MGO+AG (1.0 mM) as a positive control. ( B,C ) Quantitative data of representative cytograms of Annexin V-FITC and PI staining. Percentage of control (LL), early-stage apoptotic (LR), late-stage apoptotic (UR), and necrotic cells (UL) as analyzed using BD CellQuest Pro software. ( D ) MES13 cells were pretreated with l -cysteine for 1 h, followed by 500 μM MGO for 1 h. Green fluorescence (ROS generation) from 2′,7′-Dichlorofluorescin diacetate (DCF-DA) was examined by JuLI live-cell imaging system. Scale bar indicates 500 μm. ( E ) Quantitative measurements of fluorescent intensity were evaluated using Image J software. All data are presented as mean ± SEM. n = 3 (## p < 0.01, ### p < 0.001 vs. Control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Incubation, Flow Cytometry, Control, Positive Control, Software, Fluorescence, Live Cell Imaging

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on apoptosis-related proteins and mitogen-activated protein kinase (MAPK) signaling pathway in MES13 cells. Cells were pretreated with l -cysteine for 1 h, followed by incubation with 500 μM MGO for 24 h. ( A ) The protein expression levels of Bax, Bcl-2, Caspase-3, and PARP were measured using western blot. ( B – D ) Bax/Bcl-2 ratio, Caspase-3, and PARP band intensity; α-tubulin was used as an internal control. ( E ) MES13 cells were pretreated with l -cysteine for 1 h, then incubated with 500 μM MGO for 1 h. The protein expression levels of MAPKs (extracellular signal-regulated kinase; ERK, c-Jun N terminal kinase; JNK, p38, and phosphorylated form) were examined by western blot. ( F – H ) p-ERK/ERK, p-JNK/JNK, and p-p38/p38 ratio and intensity; α-tubulin was used as an internal control. All data are presented as mean ± SEM. n = 3 (## p < 0.01, ### p < 0.001 vs. Control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Expressing, Western Blot, Control

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on glyoxalase system-related protein and D-lactate production in MGO-induced MES13. Cells were pretreated with l -cysteine (0.1, 0.5, and 1.0 mM) for 1 h, followed by incubation with 500 μM MGO for 24 h. ( A ) The protein expression levels of Sirt1 and GLO-I were evaluated by western blot. ( B , C ) Sirt1 and GLO-I band intensity; α-tubulin was used as an internal control. ( D ) Quantitative levels of D-lactate were shown in MES13 cells. All data are presented as mean ± SEM. n = 3 (# p < 0.05, ## p < 0.01, ### p < 0.001 vs. Control, ** p < 0.01, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Incubation, Expressing, Western Blot, Control

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on MGO-induced cell toxicity and ROS generation in HEK 293 cells. ( A ) Cell viability of HEK 293 cells with MGO (500 μM) and various concentrations of l -cysteine (0.1, 0.5, and 1.0 mM) and analyzed using the MTT assay. ( B ) MGO-induced LDH production was evaluated by LDH assay in HEK 293 cells. ( C ) Representative cytograms of Annexin V-FITC and PI staining of MGO-induced HEK 293 cells. Cells were pre-treated with several concentrations l -cysteine for 1 h, then incubated with MGO (500 μM) for 24 h. After 24 h, the concentrations of viable (Annexin V-FITC and PI negative cells), early-stage apoptotic (Annexin V-FITC positive, PI negative cells), late-stage apoptotic (Annexin V-FITC positive, PI-positive cells), and necrotic (PI-positive cells) cells were analyzed by flow cytometry. ( a ) control; ( b ) 500 μM MGO; ( c ) MGO+ l -cysteine (0.1 mM); ( d ) MGO+ l -cysteine (0.5 mM); ( e ) MGO+ l -cysteine (1.0 mM); ( f ) MGO+AG (1.0 mM) as a positive control. ( D , E ) Percentage of control, early-stage apoptotic, late-stage apoptotic, and necrotic cells as analyzed by flow cytometry. ( F ) MES13 cells were pretreated with l -cysteine for 1 h, followed by 500 μM MGO for 1 h. Green fluorescence (ROS generation) from DCF-DA was examined by FAC analysis. Quantitative measurements of fluorescent intensity were evaluated using an FAC analysis system. ( a ) control vs 500 μM MGO; ( b ) 500 μM MGO+ l -cysteine (0.1 mM); ( c ) MGO+ l -cysteine (0.5 mM); ( d ) MGO+ l -cysteine (1.0 mM); ( e ) MGO+ AG (1.0 mM) as a positive control. All data are presented as mean ± SEM. n = 3 ( ### p < 0.001 vs. Control, * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: MTT Assay, Lactate Dehydrogenase Assay, Staining, Incubation, Flow Cytometry, Control, Positive Control, Fluorescence

Journal: Cells

Article Title: Effect of Cysteine on Methylglyoxal-Induced Renal Damage in Mesangial Cells

doi: 10.3390/cells9010234

Figure Lengend Snippet: Effects of l -cysteine on MGO-induced cytoskeletal proteins in MES13 cells. ( A ) MES13 cells were pretreated with l -cysteine for 1 h, followed by 500 μM MGO for 1 h. The cells were stained with Hoechst 33342 (blue) and F-actin (Red) Alexa Fluor 555 ® Phalloidin, and observed using confocal microscopy. The blue color indicated DAPI (nuclear) staining. Scale bar indicates 100 μm. ( B ) Quantitative measurements of F-actin fluorescence intensity were evaluated using NIS-Elements imaging software. Scale bar indicates 10 μm. All data are presented as mean ± SEM. n = 3 ( ## p < 0.01 vs. Control, *** p < 0.001 vs. MGO 500 μM).

Article Snippet: SV 40 MES13 (murine mesangial) and HEK 293 (human embryonic kidney) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Confocal Microscopy, Fluorescence, Imaging, Software, Control

Journal: Cell Cycle

Article Title: Specificity of calcium/calmodulin-dependent protein kinases in mouse egg activation

doi: 10.4161/cc.28432

Figure Lengend Snippet: Figure 1. CaMKIIγ and δ, but not CaMKI or CaMKIV, can trigger MII exit in mouse eggs. (A) Schematic representation of the experimental procedure. cRNAs encoding EGFP or EGFP fused to different CaM kinases were microinjected into Camk2g−/− (KO) GV oocytes. As a control, Egfp cRNA was also injected into wild-type (WT) oocytes. Indicated are the catalytic and Ca2+/CaM-binding (CaM) domains, as well as the catalytic site (K43, K49, or K75) of each CaM kinase. Injected oocytes were matured in vitro, parthenogenetically activated with 10 mM SrCl2 (Sr2+) for 3 h, and scored for activation by pronucleus formation. (B) Paired EGFP fluorescence (top) and DIC images following injection of the cRNAs depicted in (A) into Camk2g−/− (KO) or Camk2g+/+ (WT) GV oocytes. The concentration of the different CaMK constructs was adjusted to yield similar levels of GFP fluorescence 4 h after injection (upper panel). The lower panel shows the presence of pronuclei in the control and Camk2g- and Camk2d-injected eggs 16 h after activation. A representative image of 4 experiments is shown. Magnification: 200×. (C) Comparable expression levels of the different constructs. The concentration of the different CaMK constructs was adjusted to yield similar levels of GFP fluorescence upon injection. The fluorescence was quantified using ImageJ software. The experiment was conducted 4 times, and the data are expressed as the mean ± SEM; at least 6 oocytes were analyzed in each group. (D) Egg activation, as measured by pronuclear formation, in the different groups 16 h after exposure to 10 mM SrCl2. The data are expressed as the mean ± SEM. Statistical analysis was performed using ANOVA. *P < 0.0001 vs. KO eggs injected with Egfp cRNA.

Article Snippet: 30 The TaqMan gene expression assays used (Life Technologies) were as follows: Mm00437967_m1 (CaMKIIα), Mm00432296_m1 (CaMKIIβ), Mm00618054_m1 (CaMKIIγ), Mm00499266_m1 (CaMKIIδ), Mm00519436_m1 (CaMKI), and Mm01135329_m1 (CaMKIV).

Techniques: Control, Injection, Binding Assay, In Vitro, Activation Assay, Fluorescence, Concentration Assay, Construct, Expressing, Software