Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO, bars 1-4) or 660 nM TSA (TSA, bars 5-8) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of seven independent experiments. Asterisks denote significant differences (***p<0.001). NS, not significant. ## p<0.01 bar 5 vs 1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with increasing TSA doses (0, 6.6, 66, 660, 6600 nM) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of three independent experiments. Asterisks denote significant differences (p<0.05). NS, not significant. # p<0.05 vs 0 nM control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were transfected with non-targeting luciferase siRNA (Luc), or siRNA against SMRT, NCoR, or PPARγ. The levels of mRNA were determined by qPCR. Each point represents the mean ± S.E. of at least three independent experiments. (B) 3T3-L1 adipocytes were transfected with control (Luc), SMRT, NCoR, or PPARγ siRNA. 24 h post transfection, cells were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα) for additional 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01). NS, not significant.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were treated with DMSO, 660 nM TSA, 20 µM SAHA, 10 µM MS275, or 5 µM MC1568 for 24 h. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. Samples were treated in duplicate. Representative immunoblots from three independent experiments are shown. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA, 10 µM MS275, 5 µM MC1568, or combination of MS275 and MC1568 (MS275+MC1568) for 24 h. (C) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA (TSA), 5 or 20 µM SAHA (SAHA) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01; ***p<0.001). NS: not significant. # p<0.05, ## p<0.01 vs the corresponding DMSO Control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A and B) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 660 nM TSA, together with or without 1 µM Rosi (Rosi) for 24h. Cells were then treated with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western analysis with the indicated antibodies. Representative immunoblots and quantification data from five independent experiments are shown in 5A and B, respectively. (C) ERK phosphorylation correlates highly with lipolysis in the treatments of Rosi, TNFα, or both in the presence or absence of TSA, as shown by fitting with linear regression. Individual values were obtained from the experiments described in Figures. 1 and 7B.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 25 µM U0126 for 1 h, followed by treatment with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with DMSO (bars 1-4), 660 nM TSA (bars 5-8), 25 µM U0126 (bars 9-12), or both (bars 13-16) for 24h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (**: p<0.01). NS, not significant. ## p<0.01 vs Treatment No.1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced cardiomyocyte oxidative stress. (A, B) Cell viability was examined using Cell Counting Kit-8 (CCK-8) assay. (C) Klotho protein levels were examined using western blot analysis. (D) DQ-stimulated H9c2 cardiomyocytes were treated with recombinant Klotho protein, followed by viability assessment using CCK-8 assay. (E, F) Apoptosis was examined using flow cytometry. (G, H) ROS fluorescence signal, (I) MDA level, (J) GSH-ST, (K) GSH-PX, and (L) SOD activities in H9c2 cells were assessed using ELISA kits. (M, N) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. Data were presented as mean ± SD. * P <0.05, ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Cell Counting, CCK-8 Assay, Western Blot, Recombinant, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Nrf2 inhibition abrogated the protective effects of Klotho on DQ-induced oxidative stress. DQ-stimulated H9c2 cells received recombinant Klotho protein and Nrf2 inhibitor ML385. (A, B) Nrf2, HO-1, and NQO1 protein levels were examined using western blot analysis. (C) Cell viability was examined using CCK-8 assay. (D, E) Apoptosis was examined using flow cytometry. (F, G) ROS fluorescence signal, (H) MDA level, (I) GSH-ST, (J) GSH-PX, and (K) SOD activities in H9c2 cells were assessed using ELISA kits. Data were presented as mean ± SD. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group. && P <0.01, compared to DQ+Klotho group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Inhibition, Recombinant, Western Blot, CCK-8 Assay, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: American Journal of Translational Research

Article Title: Klotho mitigates diquat-induced myocardial injury in rats by activating Nrf2/ARE-mediated suppression of oxidative stress

doi: 10.62347/SKBR3572

Figure Lengend Snippet: Klotho alleviated DQ-induced acute myocardial injury in rats through Nrf2/ARE activation. Rats were intragastrically administered with DQ to induce acute myocardial injury and treated with recombinant Klotho protein for 5 days. (A, B) Klotho protein expression in myocardial tissues was assessed using immunohistochemistry. (C) H&E staining of myocardial tissue of rats in each group (D, E) ROS fluorescence signal, (F) MDA level, (G) GSH-ST, (H) GSH-PX, and (I) SOD activities in H9c2 cells were assessed using ELISA kits. (J, K) Nrf2, HO-1, and NQO1 protein levels in myocardial tissues was assessed using immunohistochemistry. ** P <0.01, compared to control group. ## P <0.01, compared to DQ group.

Article Snippet: To induce oxidative injury, cells were treated with 50 μM DQ (45422, Sigma-Aldrich, USA) for 24 h. For Klotho intervention, cells were treated with 1 μg/mL recombinant Klotho protein (1819-KL, R&D Systems, USA) [ 19 , 20 ].

Techniques: Activation Assay, Recombinant, Expressing, Immunohistochemistry, Staining, Fluorescence, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 1. RAW cells chemotax toward CX3CL1 (fractalkine). A, Live cells were incubated with rabbit anti-CX3CR1 Ab or control IgG at 4°C before fixation and incubation with anti-rabbit IgG Ab conjugated to Alexa Fluor 488. Immunofluores- cence images are shown as well as the corresponding phase contrast im- ages (see insets). Scale bar 10 m. B, Cell migration in response to in- creasing doses of CX3CL1 was mea- sured as described in Materials and Methods. C, To distinguish between chemotaxis and chemokinesis, cell migration was assessed in response to 50 ng/ml CX3CL1 added in either the bottom chamber or the top chamber of the transmigration apparatus. D, The specificity of CX3CL1-induced chemotaxis was verified by preincu- bating the cells with 10 g/ml CX3CR1 neutralizing Ab or control IgG for 1 h before subjecting the cells to the transmigration assay. E, Cells were preincubated for 5 h with or without 250 ng/ml pertussis toxin (PTX) before measuring cell migra- tion as described above. n 3. , p 0.05 compared with the correspond- ing controls.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Incubation, Control, Migration, Chemotaxis Assay, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 2. CX3CL1 treatment induces a rearrangement of the actin cytoskeleton. A, RAW cells were either untreated (Untr) or treated with 50 ng/ml CX3CL1 for 1 min before fixation and staining of F-actin using phalloidin conjugated to Alexa Fluor 568. Images are representatives of at least five independent experiments. Scale bar 10 m. B, Cells were treated for various times with CX3CL1, then fixed, and total F-actin, nor- malized to the cell number, was quantitatively measured as described in Materials and Methods. n 3–7 independent determinations for each time point.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 3. CX3CL1 stimulates tyrosine phosphorylation. A, RAW cells were treated for various times with 50 ng/ml CX3CL1, and total cell lysates were subjected to Western blotting using a phosphotyrosine-spe- cific Ab (PY) or using anti--actin Ab as a verification of equal protein loading. A Western blot representative of three independent experiments is shown. B, RAW cells were either untreated or treated with CX3CL1 for 1 min before fixation and costaining of F-actin and phosphotyrosine-contain- ing proteins. C, Murine bone marrow-derived macrophages, prepared as described in Materials and Methods, were stimulated with CX3CL1 for 1 min before costaining as shown for RAW cells. Unless otherwise noted, all images shown are z-sections collected 2.5 m above the cell-substratum interface (cell midsections) using a confocal microscope. Images noted top represent the same cells as those shown below, but were taken 5 m above the coverslip level to focus on dorsal ruffles. All images are representative of at least three independent experiments. Scale bars 10 m.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 4. Syk is activated upon CX3CL1 treatment, and piceatannol inhibits CX3CL1-induced cell migration. A, RAW cells were either un- treated () or treated () with 50 ng/ml CX3CL1 for 1 min before lysis and immunoprecipitation of Syk (IP Syk) or phosphotyrosine-containing proteins (IP PY) using specific Abs. Samples were then subjected to West- ern blotting (WB) using the indicated Abs. The phosphospecific Ab used (P-Syk) specifically recognizes activated Syk (phosphorylated on residues Y519/520). Input represents the amount of Syk present in total cell lysates for each condition. Western blots representative of three independent ex- periments are shown. B, Cells were pretreated with 50 m piceatannol (Syk inhibitor) or DMSO (vehicle) and then subjected to a transmigration assay in response to CX3CL1 as described in Materials and Methods. n 3. , p 0.05 compared with CX3CL1-induced stimulation in vehicle- treated cells.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Migration, Lysis, Immunoprecipitation, Western Blot, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 6. Cells with reduced Syk expression show impaired migration toward CX3CL1. A, Syk shRNA- treated cell migration in response to CX3CL1 was de- termined using a Transwell assay as described previ- ously and compared with NI and scr cells. n 4. , p 0.05 compared with CX3CL1-induced migration in NI cells. B, The ability of Syk shRNA and scr cells to mi- grate in response to 20 ng/ml CSF-1 was also evaluated. n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, Migration, shRNA, Transwell Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 7. CX3CL1 activation of ERK1/2 is not required for RAW cell chemotaxis. A, RAW cells were stimulated with CX3CL1 for the indicated times before Western blotting of the corresponding Triton-soluble whole cell lysates. The activation statuses of ERK1/2 (p42/44MAPK), p38MAPK, and JNK1 were assessed using phosphospecific Abs, and the corresponding total (phosphorylation-independent) levels of MAPK expression are shown below as proof of equal protein loading. B, CX3CL1-induced ERK1/2 activation levels were compared between scrambled and Syk shRNA-treated cells. Results shown were obtained with a clone exhibiting 80% Syk protein reduction. C, RAW cells were preincubated for 1 h with 30 M PD98059 (MEK inhibitor) or with DMSO (vehicle) before being subjected to a transmigration assay. PD98059 efficacy was confirmed through its ability to abolish ERK1/2 phosphorylation (see inset). For all experiments, n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Activation Assay, Chemotaxis Assay, Western Blot, Phospho-proteomics, Expressing, shRNA, Transmigration Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 8. CX3CL1-induced cytoskeletal reorganization is disrupted in cells with reduced Syk expression. A, The ability of cells to increase their F-actin content after a 1-min CX3CL1 stimulation was compared among NI, scr, and Syk shRNA-treated (sh) cells, as previously described. n 4 independent experiments using heterogeneous cell populations. , p 0.05 compared with NI cells. B, Scrambled and Syk shRNA-treated cells were either untreated or treated with CX3CL1 for 1 min, and their ability to exhibit F-actin-rich cell protrusions (ruffles) was compared using F-actin staining. Representative images of three independent experiments are shown. Scale bar 10 m. The extent of CX3CL1-induced ruffles in individual cells was scored as described previously (18); protrusion in- dexes were calculated as the average of at least 50 cells in three different experiments and expressed as a percentage of scr cells. n 3. , p 0.05 compared with scrambled.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, shRNA, Staining