Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: (A) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At indicated post-infection time-periods the medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( B ) Primary bone marrow derived macrophages (BMDM) isolated from wild-type (WT) and S100A9 knockout (KO) mice were infected with IAV (2 MOI). The medium supernatant was collected to assess levels of mouse IL-6 by ELISA. ( C ) WT and S100A9 KO BMDM were infected with IAV (2 MOI). At the indicated post-infection time-period, medium supernatant was collected to assess levels of mouse TNF-α(TNF) by ELISA. ( D ) S100A9 KO BMDMs were infected with IAV (2 MOI) in the presence of purified recombinant mouse S100A9 protein (5 µg/ml). Medium supernatant was collected from infected cells to assess levels of mouse TNF and IL-6 by ELISA. Vehicle control cells (veh) were incubated with HBSS buffer. The values represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay, Isolation, Knock-Out, Purification, Recombinant, Incubation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Mouse J774A.1 macrophages were incubated with purified recombinant mouse S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic state of these cells was examined by FACS analysis of annexin V and PI stained cells. Apoptosis rate (% apoptosis) was calculated based on number of annexin V positive/PI negative cells (denoting early apoptosis)+number of annexin V positive/PI positive cells (denoting late apoptosis)/total number of cells. ( B ) Mouse alveolar macrophage MH-S cell-line was incubated with purified S100A9 protein (5 µg/ml) for 48 h and 72 h. The apoptotic status was determined as described in (A). ( C ) Mouse J774A.1 macrophages were infected with IAV (2 MOI) in the presence of either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab). At 48 h post-infection, the apoptotic state of these cells was determined as described in (A). The values (i.e. annexin V and PI staining quantified by FACS) represents mean ± standard deviation from three independent experiments, *p<0.05 by Student's t test. Veh; cells incubated with HBSS buffer (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Incubation, Purification, Recombinant, Staining, Infection, Blocking Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Survival of IAV infected (1×10 5 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The data represents values from two independent experiments performed with 5 mice/group for each experiment (total 10 mice/group from two experiments); *p = 0.03. ( B ) Hematoxylin and eosin (H&E) staining of lung sections from mock infected or IAV infected mice (3×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or S100A9 Ab (24 h prior to IAV inoculation, 2 mg of antibody/mouse was administered via i.p route). Magnification, ×10. ( C ) Mice were administered with purified recombinant mouse S100A9 protein (15 µg/mouse) via intra-tracheal route. At 8 h post-administration, levels of mouse TNF-α in the lung was assessed by performing ELISA analysis with lung homogenate. ( D ) Lung homogenate prepared from mock infected and IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route) were subjected to ELISA analysis to determine levels of mouse TNF-α in the lung. ( E ) For ex-vivo experiment, broncho-alveolar lavage fluid (BALF) was collected (at 3 d post-infection) from IAV infected mice (2×10 4 pfu/mouse via intra-tracheal route) administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). The BALF cells were isolated and plated in 48-well plate. After 2 h and 4 h, the medium supernatant was analyzed for mouse TNF-α (TNF) and mouse IL-6 by ELISA. Values shown in (C), (D) and (E) represent the mean ± standard deviation from three independent experiments performed in triplicate. *p<0.05 using a Student's t test. Veh; HBSS buffer diluted in PBS (vehicle control).

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, Staining, Purification, Recombinant, Enzyme-linked Immunosorbent Assay, Ex Vivo, Isolation, Standard Deviation

Journal: PLoS Pathogens

Article Title: DAMP Molecule S100A9 Acts as a Molecular Pattern to Enhance Inflammation during Influenza A Virus Infection: Role of DDX21-TRIF-TLR4-MyD88 Pathway

doi: 10.1371/journal.ppat.1003848

Figure Lengend Snippet: ( A ) Lung sections were prepared (at 3 d post-infection) from IAV infected (2×10 4 pfu/mouse via intra-tracheal route) mice administered with either control IgG (IgG) or anti-S100A9 blocking (neutralizing) antibody (S100A9 Ab) (24 h prior to IAV inoculation, 2 mg of antibody/mouse administered via i.p route). For each experimental group lung sections were prepared from three control IgG treated mice (+IAV) and three S100A9 Ab treated mice (+IAV). The lung sections were used for TUNEL staining. Image J software was used to calculate TUNEL-positive areas (representing apoptosis) in the lung sections as detailed in the methods section. The data is presented as percent apoptotic area. The percent apoptotic area was calculated from nine areas/lung section as detailed in the methods section. The values were compiled to calculate the percent apoptotic area in IAV infected IgG treated mice vs. IAV infected S100A9 Ab treated mice, * p = 0.0164 by Student's t test. ( B ) A representative TUNEL staining of lung sections from IAV infected mice administered with either IgG or S100A9 Ab. The apoptotic nuclei (representing apoptosis) are indicated with red arrows. ( C ) A schematic model depicting the role of extracellular S100A9 and DDX21/TRIF/S100A9/TLR4/MyD88 signaling network in exaggerating lung disease during IAV infection. PM, plasma membrane; NM, nuclear membrane.

Article Snippet: The plates were washed three times with PBST and incubated with either goat anti-mouse IgG (300 ng/well) (R&D) (for mouse S100A9) or mouse anti-human IgG (50 ng/well) (R&D) (for human S100A9) in PBST+0.1% BSA for 2 h at room temperature.

Techniques: Infection, Blocking Assay, TUNEL Assay, Staining, Software

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Single S100A8 or S100A9 yeast transformants or (B) cotransformants with plasmids mCherry S100A8/ GFP S100A9 were grown overnight in SG, and images were obtained with a fluorescence microscope. (C) TCA precipitates of extracts from cells growing on glucose or galactose medium were separated by 10% SDS-PAGE and analyzed by Western blot.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Fluorescence, Microscopy, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Fluorescent microscope images of GFP S100A8 and GFP S100A9 (green) after (A) 2 days or (B) 4 days of induction. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (C) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole or in the cytoplasm, following 2 and 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: GFP (green) and mCherry (red) fluorescent microscope images of mCherry S100A8/ GFP S100A9 cotransformed cells after 2 or 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Confocal images of GFP, GFP S100A8 or GFP S100A9 (green) after 2 days of induction in pep4Δ mutants cells. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (B) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole of pep4 Δ cells, following 2 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques:

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) NI, noninduced control (Lanes 1, 4, 7, 10). Extracts of cells induced for 2 or 4 days to produce GFP S100A8 (Lanes 2 and 3), GFP S100A9 (Lanes 5 and 6) and mCherry S100A8/ GFP S100A9 (Lanes 8, 9 and 11,12) were separated on a native gel and analyzed by Western blot. (B) Semi-denaturing agarose detergent gel. After 2 days of induction GFP S100A8 (Lane 1), GFP S100A9 (Lane 3) or cotransformants mCherry S100A8/ GFP S100A9 (Lanes 7, 8 and 10, 11) formed SDS-stable aggregates in yeast cells. Boiling (+) the samples led to full soloubilization of aggregates to the monomeric form (Lanes 2 and 4). Total cell extracts (180 µg) were resolved using SDD-AGE. Blots were probed with anti-GFP or mCherry antibodies. Total cell extract of Q103 GFP cells (90 µg) was prepared after 24 h of induction (lane 5). (C) Filter retardation assay of cells grown for 3 and 5 days under inducing conditions. Loading control was visualized by CBB staining. Empty vector-transfected cells were used as control. (D) Spheroplasts of control and induced cells stained with ThT after 3 days of incubation on galactose plates.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Control, Western Blot, Staining, Plasmid Preparation, Transfection, Incubation

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Ten-fold dilutions of yeast cells transformed with p GFP-S100A8 , p GFP-S100A9 or both plasmids were plated on glucose (non-inducing) or galactose (inducing) plates and photographed after 72 h. (B) Non-tagged proteins as in A.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) wild type and cdc53-1 ts mutant cells expressing p YES2-S100A8 or p YES2-S100A9 were spotted on glucose or galactose plates and photographed after 72 h. (B ) Semi-denaturing agarose gel. After 2 days of induction GFP S100A9 forms aggregates in wild type and ts strain cdc53-1 at 30°C and 32°C. Total cell extracts (180 µg) were resolved using SDD-AGE. (C ) Ten-fold dilutions of cdc53-1 , cdc34-2 , srp1-31, and sec27-1 yeast cells transformed with p TET-S100A8 or p TET-S100A9 were spotted on SD plates with (inducing) or without (non-inducing) 5 µg/ml doxycycline and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Expressing, Agarose Gel Electrophoresis, Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Viability of wild type or hsp104Δ yeast in the presence of S100A8, S100A9 and S100A8/9 proteins. p YES2-S100A8 , p YES2-S100A9 or both plasmids were expressed in wild type or hsp104Δ mutant cells. Viability was monitored using the spot test assay on inducing (galactose) or noninducing (glucose) plates. (B) Ten-fold dilutions of wild type cells or hsp104Δ mutants transformed with p GALSc104(WT) and with p YES2-S100A8, p YES2-S100A9 or both S100 plasmids were plated on glucose (non-inducing) or galactose (inducing) plates. (C) Confocal images of GFP S100A8 and GFP S100A9 after 2 days of induction in wild type or Δhsp104 mutant cells. (D) Cell extracts were prepared from wild type or hsp104Δ mutant cells expressing p GFP-S100A8 or p GFP-S100A9 after 2 days of induction. Extracts were incubated in 2% SDS sample buffer with (+) or without (−) boiling, loaded on agarose gels, and analyzed by Western blot using anti-GFP antibodies to detect the S100A8 and S100A9 proteins.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Spot Test, Transformation Assay, Expressing, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Cells expressing empty vector (p YES2), p YES2-S100A8 , p YES2-S100A9 , or cotransformants with p YES2-S100A8/ p YES2-S100A9 in sse1, sse2, ssa1, ssa2, ssa3, hsp26, and ydj1 mutants and the isogenic wild type parent were spotted on galactose and glucose plates and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Expressing, Plasmid Preparation