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: EMBO Molecular Medicine

Article Title: An immunohistochemical atlas of necroptotic pathway expression

doi: 10.1038/s44321-024-00074-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: For experiments in Fig. , co-housed 8-week-old female C57BL/6J wild-type mice were administered either 300 μg/kg TNF (R&D Systems Cat#410-MT/CF Lot CS152103 ) in endotoxin-free DPBS or endotoxin-free DPBS alone (Merck Cat# TMS-012-A) via bolus tail vein injection.

Techniques: Derivative Assay, Polymer, Plasmid Preparation, Blocking Assay, Membrane, Staining, RNA HS Assay, dsDNA Assay, Protease Inhibitor, Software, Imaging, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Effect of microglial Pd1 on glial scar formation after spinal cord injury in mice

doi: 10.1016/j.jbc.2025.108489

Figure Lengend Snippet: Microglia was involved in the functional recovery after SCI by regulating inflammatory response . A , immunofluorescence staining of Iba1 was conducted on the spinal cords of mice in sham group, control group, SCI + saline group and SCI + minocycline group. The sham group's mice underwent laminectomy without SCI and were sampled concurrently with the other groups. The scale bar represented 100 μm. B , quantification of Iba1 immunostaining intensity in the spinal cords of mice in sham group, control group, SCI + saline group, and SCI + minocycline group. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, as determined by one-way ANOVA. B : one-way ANOVA, F (3, 16) = 8.570, p = 0.0013; sham versus control, p = 0.0112; sham versus SCI + saline, p = 0.0012; SCI + saline versus SCI + minocycline, p = 0.0442; n = 5 mice per group). C , locomotor recovery evaluated by the BMS score acquired at different time points after SCI in mice treated with nothing, saline, or minocycline. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, as determined by two-way ANOVA. C : two-way ANOVA, F (12, 80) = 29.89, p < 0.0001; 7 days, SCI + saline versus SCI + minocycline, p = 0.0017; 14 days, SCI + saline versus SCI + minocycline, p = 0.0099; n = 6 mice per group). D – G , minocycline treatment reduced SCI-induced IL-6 and TNF-α mRNA expression and increased IL-10 mRNA expression in the injured spinal cord. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by one-way ANOVA. D : one-way ANOVA, F (3, 16) = 13.27, p = 0.0001; sham versus control, p = 0.0078; sham versus SCI + saline, p = 0.0002; SCI + saline versus SCI + minocycline, p = 0.0022; n = 5 mice per group) E : one-way ANOVA, F (3, 16) = 2.660, p = 0.0834; n = 5 mice per group) F : one-way ANOVA, F (3, 15) = 13.48, p = 0.0002; sham versus control, p = 0.0013; sham versus SCI + saline, p = 0.0002; sham versus SCI + minocycline, p = 0.0014; control versus SCI + minocycline, p = 0.0001; n = 4–5 mice per group) G : one-way ANOVA, F (3, 16) = 26.44, p < 0.0001; sham versus control, p < 0.0001; sham versus SCI + saline, p = 0.0001; control versus SCI + minocycline, p < 0.0001; SCI + saline versus SCI + minocycline, p < 0.0001; n = 5 mice per group). H , locomotor recovery evaluated by the BMS score acquired at different time points after SCI in mice treated with nothing, saline, minocycline, minocycline (+TNF-α), minocycline (+IL-6), and minocycline (+IL-10 neutralizing antibody). All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by two-way ANOVA. H : two-way ANOVA, F (18, 134) = 1.336, p = 0.1754; 14 days, SCI + minocycline versus SCI + minocycline (+TNF-a), p = 0.0002; SCI + minocycline versus SCI + minocycline (+IL-6), p = 0.0014; SCI + minocycline versus SCI + minocycline (+IL-10 neutralizing antibody), p = 0.0160; n = 5 to 6 mice per group). BMS, Basso Mouse Scale; IL-6, interleukin-6; SCI, spinal cord injury; TNF-α, tumor necrosis factor-alpha.

Article Snippet: TNF-α (Novus, NBP2-35185), IL-6 (MCE, HY-P7063), and IL-10 neutralizing antibody (MCE, HY-P990001) were applied at doses of 10 ng, 10 ng, and 2 μg, respectively, dissolved in 2 μl of minocycline solution.

Techniques: Functional Assay, Immunofluorescence, Staining, Control, Saline, Immunostaining, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Effect of microglial Pd1 on glial scar formation after spinal cord injury in mice

doi: 10.1016/j.jbc.2025.108489

Figure Lengend Snippet: Cytokine array analysis revealed Pd1-dependent cytokines and chemokines in LPS-treated microglia . A , qPCR showing the decreased Pd1 mRNA in the microglia after Pd1 siRNA (20 nM and 50 nM) treatment for 48 h. All data are presented as mean ± SD. Statistical significance was indicated by ∗∗ p < 0.01, ∗∗∗ p < 0.001, as determined by one-way ANOVA. A : ANOVA, F (2, 12) = 15.32, p = 0.0005; NC siRNA versus Pd1 siRNA 20 nM, p = 0.0022; NC siRNA versus . Pd1 siRNA 50 nM, p = 0.0004; n = 5 wells per group). B , array membranes of protein expression among NC siRNA, NC siRNA + LPS (5 μg/ml), Pd1 siRNA (50 nM), and Pd1 siRNA (50 nM) + LPS (5 μg/ml) groups in culture medium for microglia. C , the expression of one colony-stimulating factor (G-CSF), one intercellular cell adhesion molecule (CD54), two inflammatory factors (IL-6 and TNF-α), and four chemokines (CXCL10, CCL12, CXCL9, and CCL5) significantly increased following LPS treatment, while the expression of one chemokine (CCL4) decreased. A significant increase in the expression of one inflammatory factor (TNF-α) and one chemokine (CXCL9) after treatment with Pd1 siRNA + LPS. All data are presented as mean ± SD. Statistical significance was indicated by ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, as determined by two-way ANOVA. (Figure 6C: two-way ANOVA; G-CSF, NC siRNA versus Pd1 siRNA + LPS, p = 0.0016, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0021; CD54, NC siRNA versus Pd1 siRNA, p = 0.0245, NC siRNA versus Pd1 siRNA + LPS, p = 0.0185; IL-6, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001; Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; CXCL10, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0481; CCL12, NC siRNA versus NC siRNA + LPS, p = 0.0012, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017, NC siRNA + LPS versus Pd1 siRNA, p = 0.0012; Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0017; CXCL9, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0156, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0145; CCL4, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA, p = 0.0062, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p = 0.0143; CXCL2, NC siRNA versus NC siRNA + LPS, p = 0.0121, NC siRNA versus Pd1 siRNA, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p = 0.0017; CCL5, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; TNF-α, NC siRNA versus NC siRNA + LPS, p < 0.0001, NC siRNA versus Pd1 siRNA + LPS, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA, p < 0.0001, NC siRNA + LPS versus Pd1 siRNA + LPS, p = 0.0442, Pd1 siRNA versus Pd1 siRNA + LPS, p < 0.0001; n = 3–4 separate experiments per group). G-CSF, granulocyte colony-stimulating factor; IL-6, interleukin-6; LPS, lipopolysaccharide; NC, negative control; Pd1, programmed cell death protein 1; qPCR, quantitative real-time PCR; siRNA, small interfering RNA; TNF-α, tumor necrosis factor-alpha.

Article Snippet: TNF-α (Novus, NBP2-35185), IL-6 (MCE, HY-P7063), and IL-10 neutralizing antibody (MCE, HY-P990001) were applied at doses of 10 ng, 10 ng, and 2 μg, respectively, dissolved in 2 μl of minocycline solution.

Techniques: Expressing, Negative Control, Real-time Polymerase Chain Reaction, Small Interfering RNA